ES2580161T3 - Soft gel systems in the modulation of stem cell development - Google Patents

Abstract

A method of inducing the differentiation of a population of quiescent mesenchymal stem cells in a population of a type of cell of interest, said method comprising the steps of (a) maintaining said population of quiescent mesenchymal stem cells, while retaining the ability to said population of mesenchymal stem cells is differentiated into multiple cell types and preserving the proliferative capacity of said population of mesenchymal stem cells in the presence of an apparatus containing a gel or a gel matrix having a shear module in a range of 150 -750 Pa; and (b) culturing said mesenchymal stem cell population in the presence of an induction medium, thereby inducing differentiation of said mesenchymal stem cell population into a population of a cell type of interest.

Description

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DESCRIPTION

Soft gel systems in the modulation of stem cell development Field of the invention

The present invention provides methods of modulating the development of stem cells using soft gels. Specifically, the invention provides methods for modulating the development of stem cells, using gels that have optimized viscoelastic properties.

Background of the invention

Adult mesenchymal stem cells have the ability to self-renew and differentiate into multiple cell lineages of mesenchymal tissues. Therefore, we have taken into account the chemical applications of these cells, such as replacement of damaged tissues or vines for antineoplastic agents. At this time, the applications of adult mesenchymal stem cells are still limited to a pre-thermal state, partly due to the rapid aging of these ex vivo cells, which limits their expansion and technical modification. It has been indicated that immortalization of mesenchymal stem cells through telomerase transduction overcomes problems associated with accelerated aging. However, their capacity for unlimited self-renewal can lead to out of control growth, once they have been implanted in tissues. In fact, in vitro environments, transformation of mesenchymal stem cells transduced with telomerase was observed.

Therefore, the regulation of the growth of adult mesenchymal stem cells is one of the key stages for its chemical applications.

Engler et al Cell Vol. 126, No. 4, pages 677-689 (2006) show that the elasticity of the matrix leads to the specification of the stem cell lineage. The mesenchymal stem cells (CMM) vfrgenes showed that they specified lineage and were dedicated to phenotypes with extreme sensitivity to elasticity at the tissue level.

Zhou et al, Chinese Journal of Cellular and Molecular Immunology 23 (4), pages 369-371 (2007) refer to the effect of low serum culture on the cell cycle syncytium of mesenchymal stem cells. CMCs were cultured and identified with CD44, CD90, CD71 and CB11b by flow cytometna. The cell cycle and apoptosis were detected in normal and low serum culture by flow cytometna. It was found that prolonged culture in serum deprived medium induced the arrest of the cell cycle in the G0 / G1 stage.

Summary of the invention

The present invention provides methods of modulating the development of stem cells using soft gels. Specifically, the invention provides methods of modulating the development of stem cells, using gels that have optimized viscoelastic properties.

The present invention provides a method of inducing the differentiation of a population of quiescent mesenchymal stem cells in a population of a type of cell of interest, said method comprising the steps of (a) maintaining said population of quiescent mesenchymal stem cells, preserving the ability of said population of mesenchymal stem cells to be differentiated into multiple types of cells and preserving the proliferative capacity of said population of mesenchymal stem cells in the presence of an apparatus containing a gel or a gel matrix having a shear modulus in a range of 150-750 Pa; and (b) cultivate said

population of mesenchymal stem cells in the presence of an induction medium, thereby inducing the

differentiation of said population of mesenchymal stem cells in a population of a type of cell of interest.

In one embodiment, the population of a type of cell of interest is a population of adipocytes.

In one embodiment, the population of a type of cell of interest is a population of osteoblasts.

In some embodiments:

(a) the gel or gel matrix further comprises an animal serum; I

(b) the stage of maintenance of the population of mesenchymal stem cells in a gel or a gel matrix is preceded by a culture stage of the population of mesenchymal stem cells in a tissue culture apparatus; I

(c) the maintenance stage is performed directly after the isolation, purification or enrichment of the population of mesenchymal stem cells from a biological sample.

In one embodiment, the induction medium is an adipogenic induction medium.

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In some embodiments:

(a) the gel or gel matrix comprises acrylamide and bisacrylamide; I

(b) the gel or gel matrix is two-dimensional or three-dimensional; I

(c) the gel or gel matrix is coated with an adhesion protein; I

(d) the population of mesenchymal stem cells is a population of adult mesenchymal stem cells; I

(e) The population of mesenchymal stem cells is a population of human mesenchymal stem cells.

In some embodiments:

(a) the gel or gel in the matrix has a total acrylamide concentration of 3% and a total bisacrylamide concentration of 0.06% to 0.5%, a total acrylamide concentration of 5.5% and a concentration total bisacrylamide from 0.05% to 0.075% or a total acrylamide concentration of 7.5% and a total bisacrylamide concentration from 0.01% to 0.03%; I

(b) the adhesion protein is a collagen, a fibronectin, or a combination thereof.

Brief description of the figures

Figure 1. A. Mechanical properties of polyacrylamide substrates. The shear modulus of polyacrylamide gels was measured with a range of proportions of acrylamide (indicated as percentages near the data lines) to bisacrylamide (indicated as crosslinking agent). The shear module (G '), expressed in Pascals, increases at constant polymer mass with increasing crosslinking agent. The increase in acrylamide concentration from 3 to 12% also creates a large stiffness range of 10 to 50,000 Pa. The continuous line indicates the theoretical stiffness of a rubber type network if each cross-linking was elastically effective. B. Cell shape and structure of CMMh F-actin in hard or soft matrices. C. Cell shape and structure of CMMh F-actin in soft gels and glass.

Figure 2. Incorporation of BrdU in CMMh.

Figure 3. The effect of matrix stiffness on adipocyte differentiation. A. Percentage chart of positive cells. First bar in each series: Staining with Oil Red O. Second bar: staining of PPARy2.

Figure 4. Structure of F-actin in astrocytes seeded in nigid or soft gels.

Figure 5. Quantification of the increase in Rho activity of soft gels with respect to hard gels. Astrocytes were seeded in polyacrylamide gels with various stiffnesses. Rho's GTP load level was quantified.

Figure 6. Melanoma cells spread more in nested matrices. Graphic representation of area.

Figure 7. Melanoma cells adhere to soft and neat gels with the same efficiency.

Figure 8. Largest population of melanoma cells in nid gels.

Figure 9 shows images of human CMM in various substrates of different elasticities according to various embodiments of the present invention.

Figure 10 shows the amount of bromodeoxyuridine (BrdU) captured in human CMM in substrates of various elasticities according to various embodiments of the invention.

Figure 11 shows (A-D) an illustration of the effect of an almost three-dimensional environment on the shape and proliferation of stem cells according to various embodiments of the invention.

Figure 12 shows the response of human CMC to adipogenic induction medium according to various embodiments in the invention.

Figure 13 shows the deposition of calcium visualized with Alizarin Red S after stimulation of human CMM with osteoinduction medium according to various embodiments of the invention.

Figure 14 shows a flow chart for preparing a system to induce quiescence, differentiation and proliferation in adult stem cells according to various embodiments of the invention.

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Figure 15 shows schematic illustrations of embodiments of systems of the present invention.

Detailed description of the invention

This document describes gels and matrices of gels that have a stiffness in the range of 0.01-50 kPa, methods for manufacturing it and methods for preserving a population of mesenchymal stem cells or studying mesenchymal stem cells that comprise them.

Also described herein is a method of manufacturing a polyacrylamide gel with a stiffness in the range of 150-750 Pa, which comprises the steps of polymerizing a composition comprising acrylamide and bisacrylamide, thereby producing a polyacrylamide gel. soft and coating the soft polyacrylamide gel with a composition comprising a type I collagen and a fibronectin, thereby manufacturing a polyacrylamide gel having a stiffness in the range of 150-750 Pa. In another embodiment, the composition has a mixture of acrylamide: bisacrylamide ratio between 100: 1 and 30: 1. In another embodiment, the gel has an acrylamide: bisacrylamide mixture ratio between 100: 1 and 30: 1. In another embodiment, the composition has a total acrylamide concentration of 3-5%. In another embodiment, the gel or gel matrix has a total acrylamide concentration of 3-5%. In another embodiment, the composition is a solution. In another embodiment, the composition is a suspension. In another embodiment, the composition is any other type of composition known in the art. Each possibility represents a different embodiment.

Also described herein is a method of manufacturing a fibrin matrix with a stiffness in a range of 150-750 Pa, which comprises the steps of polymerizing a composition comprising a fibrin or fibrinogen protein, thereby producing a soft fibrin matrix, in which the concentration of the fibrin or fibrinogen protein in the soft fibrin matrix is 3-10 mg / ml, and coat the soft fibrin matrix with a composition comprising an adhesion protein, manufacturing thus a fibrin matrix that has a stiffness in a range of 150-750 Pa.

This method also describes a method to preserve a population of mesenchymal stem cells, the method comprising the step of cultivating the population of mesenchymal stem cells in a gel or gel matrix with a stiffness in a range of 150-750 Pa, thus preserving a population of mesenchymal stem cells. In another embodiment, the cultivation stage is performed in the absence of chemical induction. In another embodiment, the cultivation stage is performed in the absence of an induction medium. Each possibility represents a different embodiment.

This document also describes a method to preserve a mesenchymal stem cell, the method comprising the step of culturing the population of mesenchymal stem cells in a gel or gel matrix with a stiffness in a range of 150-750 Pa, preserving in this way a mesenchymal stem cell. In another embodiment, the cultivation stage is performed in the absence of chemical induction. In another embodiment, the cultivation stage is performed in the absence of an induction medium. Each possibility represents a different embodiment.

This method also describes a method to induce the quiescence of a transformed cell, which comprises the step of culturing the transformed cell in a gel or gel matrix of the methods of the present invention, thereby inducing the quiescence of a transformed cell. In another embodiment, the transformed cell is a cancerous cell. In another embodiment, the transformed cell is a neoplasic cell. In another embodiment, the transformed cell is any other type of transformed cell known in the art. Each possibility represents a different embodiment.

In another embodiment of the methods of the present invention, the telomere length of the mesenchymal stem cell population is maintained. "Staying" refers, in another embodiment, to a lack of substantial change in length. In another embodiment, the term refers to a lack of measurable change in length. In another embodiment, the term refers to a lack of sufficient change in length that affects proliferative capacity. Each possibility represents a different embodiment of the present invention.

In another embodiment of the methods of the present invention, the population of mesenchymal stem cells is maintained in a quiescent state. "Quiescent" refers, in another embodiment, to a lack of significant replication. In another embodiment, the term refers to a large percentage of cells arrested in the cell cycle. In another embodiment, the cells stop in the G1 phase. In another embodiment, the cells stop in the G2 phase. In another embodiment, "quiescence" refers to any other definition of the term accepted in the art. Each possibility represents a different embodiment of the present invention.

In one embodiment, when the stem cell is a human mesenchymal cell derived from bone marrow, the extracellular matrix (ECM) has an elasticity of about 250 Pa and comprises a mixture of collagen and fibronectin. In another embodiment, the collagen is rat tail collagen, and the fibronectin is human fibronectin. The ratio of collagen and fibronectin can vary, and in one embodiment, the ratio of collagen to fibronectin is about 5: 1. Other collagen and fibronectin ratios can also be used. A person skilled in the art will appreciate that collagen and fibronectin can be obtained from other sources, and that

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substances other than collagen and fibronectin can be used to present elasticity and bind with integrins on the surface of the cell membrane so that the quiescence of the cell is induced.

In accordance with embodiments of the present invention, the extracellular material (MEC) is provided with an appropriate apparent elasticity by coupling the MEC with a substrate such that a stem cell comes into contact with the MEC detects the elasticity of the substrate. Consequently, the substrate can be a material whose elasticity, when coupled to the MEC, detects a stem cell that comes into contact with the MEC. In some embodiments, the substrate is glass. In other embodiments, the substrate is a gel with an elasticity of 250 Pa, or a gel with an elasticity of 7,500. These gels can be polyacrylamide gels, and, as those skilled in the art know, the elasticity of polyacrylamide gels can be modified, for example, by changing the acrylamide and bisacrylamide concentrations in the gel formulation. The manufacture of gels of different elasticity that can be used in the method of the present invention will be obvious to one skilled in the art in light of the present specification.

The elasticity of the in vivo biological environment of a stem cell can be determined by extracting a sample of physiological tissue from the immediate in vivo environment of the stem cell, and then measuring the shear modulus of that tissue sample. Exemplary procedures for preparing and measuring the elasticity of rat tissue and bovine tissue are described herein. The following table 1 provides the elasticity of various types of fabrics:

Table 1

 Species

 Elasticity Fabric (in Pa) (Mean ± DT)

 Bovine

 Bone marrow 220 ± 50

 Rat

 Subcutaneous fat 160 ± 70

 Rat

 Visceral fat 130 ± 70

 Rat

 Liver 403 ± 28

 Rat

 Skeletal muscle 2251 ± 166

In one embodiment, human CMMs in gels of 250 Pa are in a quiescent state waiting for an additional signal to determine their fate. In one embodiment, the CMMh will experience adipogenic differentiation (induced by chemical factors), or in other embodiments, a return to the cell cycle (induced by the coupling of the cells with a nidized surface), or osteogenic differentiation (which seems to require both chemical induction as a nidized substrate). Stimulation of cells grown in soft gels with adipogenic differentiation factors results in a remarkably high number of cells that accumulate drops of lipids. In one embodiment, chemical induction is required for osteoblast differentiation. The requirement of synchronized mechanical and chemical stimulation explains, in one embodiment, how human CMMs can be compartmentalized in compatible tissues, such as bone marrow, and still resist spontaneous differentiation.

As the elasticity of the matrix, in another embodiment, the choice of extracellular ligand greatly affects the adhesion and differentiation of human CMM. Type I collagen is found in a variety of tissues including bone and adipose tissue, and is regularly used as a substrate for cell adhesion experiments. In one embodiment, in 250 Pa gel, the collagen alone does not guarantee the effective adhesion of a mayonnaise of cells. In one embodiment, a mixture of type I collagen and fibronectin at a 10: 1 ratio provides the best adhesion of the cells to the 250 Pa gels without affecting the potential for differentiation.

Human CMMs have the ability to reshape their microenvironment by altering the expression of matrix metalloproteases and this helps in one embodiment to promote effective differentiation after obtaining a strong initial adhesion.

In one embodiment, the synthesis of human CMM DNA is drastically reduced when they are grown in soft gels, developing a round phenotype. This differs from other types of proliferative cells such as NIH 3T3 fibroblasts, bovine aortic endothelial cells and NRK epithelial cells that continue to divide all when grown in soft gels. Therefore, quiescence of stem cells in gels of 250 Pa is not a general induced cytokinesis insufficiency, but rather is a specific sensitivity to these cells to the substrate conformity. Accordingly, a method of maintaining stem cells in a quiescent state is described herein, which comprises suspending the stem cells in a fibronectin / collagen gel having G 'of 250 Pa.

In one embodiment, when non-proliferative human CMMs are presented with a glass substrate coated with protein gel matrix, the cells develop a fusiform morphology and re-enter the cell cycle. In another embodiment, the presence of a nested substrate cancels the physical signals of a compliant matrix. In one embodiment,

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There is no significant population of cells showing a neuronal phenotype with neurite-like bumps on soft gels of 250 Pa without any chemical induction.

In one embodiment, the elasticity of the substrate regulates the differentiation of cells with specific phenotypes. In another embodiment, the mechanical properties alone do not direct the differentiation of stem cells. This is because several tissues in the body have similar elasticities. For example, all brain, fatty and bone marrow tissues have a storage module of approximately 200 Pa, although all maintain unique cell populations. In one embodiment, human CMMs are stored in vivo in the bone marrow of an individual for decades and still retain multipotentiality. In one embodiment, human CMMs are cultured ex vivo in plastic tissue culture and retain multipotentiality for several countries. In one embodiment, both mechanical and chemical stimuli are integrated by the cell to determine its response. In another embodiment, although chemical stimuli can nullify the influences of substrate mechanics, in other embodiments, an inappropriate mechanical environment avoids a normal cellular response to chemical agonists. In one embodiment, the quiescent cells differ in osteoblasts only as a result of changing their physical and chemical environment to those that stimulate osteogenesis. Accordingly and in one embodiment, a matrix with appropriate elasticity has the ability to maintain a quiescent population of multipotential bone marrow mesenchymal stem cells that respond to both mechanical and chemical stimuli that drive proliferation and differentiation.

Figure 14 illustrates a flow chart for preparing an embodiment of a system for inducing quiescence, differentiation and proliferation in adult stem cells according to various embodiments of the present invention. Acrylamide and bisacrylamide solutions are prepared in phosphate buffered saline solution (PBS) to a total volume of 500 pl. In one embodiment, adjusting the acrylamide and bisacrylamide concentration allows a wide range of stiffness to be obtained. The polymerization is initiated with TEMED (N, N, N ', N'-tetramethylenediamine) and ammonium persulfate to form a gel. In step 601, acrylamide / bisacrylamide solution (polyacrylamide), a drop (for example, approximately 200 pl) of the polymerized gel is deposited on a glass slide previously modified with 3- aminopropyltrimethoxysilane and glutaraldehyde. In step 602, coated with N-hydroxysuccinimide in toluene, approximately 15 pl of 2% acrylic acid N-hydroxysuccinimide ester in toluene are applied to the solution of step 601 and, in step 603, top coverslips, a coverslip Chlorosilanized is placed over the drop. In step 604, removal of coverslips, the upper coverslip is removed after polymerization is completed and, optionally, the gel is illuminated with ultraviolet light for approximately 10-15 minutes (not shown). In step 605, MEC ligand, N-succinimide acrylate is reacted on top of the gel with an extracellular matrix ligand, which in one embodiment is a 0.1 mg / ml mixture of type I collagen and 0.02 mg / ml fibronectin. In an additional step (not shown), the gels are washed three times with PBS and left in PBS until step 606, gel cells, when stem cells are seeded in the cells. When mesenchymal stem cells derived from bone marrow are planted in this material, the cells become quiescent even in the presence of chemical stimuli that cause proliferation or differentiation.

Accordingly, a method is described herein to induce or maintain quiescence and sustain biological activity in an ex vivo somatic stem cell comprising: contacting the somatic stem cell with a gel matrix comprising an extracellular material that binds with integrin in the membrane of the somatic stem cell; said gel matrix having an elasticity substantially similar to the elasticity of the predominant biological microenvironment in vivo of the somatic stem cell of the same type in vivo; and provide the somatic stem cell with nutrient material to support the biological activity of the somatic stem cell ex vivo.

In a method of the present invention, a stem cell can contact appropriate MEC in various ways. For example, as described herein, the MEC can form a layer coupled to the substrate, and the stem cell can be placed in the MEC. Alternatively, the cell can be placed in MEC coupled to the substrate and additionally contacted with MEC placed in the cell, for example, by placing in the cell a structure that couples MEC with a substrate that has the apparent elasticity appropriate for the stem cell.

In another embodiment, there may be two MEC formulations: a first formulation, which may or may not include nutrient materials, that are coupled with the substrate; and a second formulation that includes nutrient materials and that do not mate with the substrate. Structures and configurations for contacting stem cells with an appropriate MEC (including substrates and, optionally, binding materials for joining the substrate with the MEC) are described herein, including, for example, Figure 6, and are obvious to a person skilled in the art in light of the present specification.

In embodiments of the methods of the present invention, a stem cell that is not in a quiescent state is contacted with MEC in accordance with methods of the present invention such that quiescence is induced in the cell and passes from a state not quiescent to a quiescent state. In other embodiments, a quiescent stem cell is contacted with MEC according to methods of the present invention such that the quiescence is maintained in the cell and does not pass into a quiescent state.

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Accordingly, a method for modulating the development of a mesenchymal stem cell is described herein, comprising the step of suspending the mesenchymal stem cell in a gel matrix comprising a gelling agent in which said gel matrix is coated. with a type I collagen, a fibronectin, or a combination thereof and wherein said gel matrix is maintained at a predetermined stiffness; and exposing the gel matrix to a modulating growth factor, whereby exposure to the chemical or physical factor results in an increase in the stiffness of the gel matrix that coincides with the stiffness of the MEC in the microenvironment in which It is intended to differentiate the mesenchymal stem cell.

In another embodiment, more than 80% of the cells are detained in the cell cycle. In another embodiment, at least 80% of the cells are detained in the cell cycle. In another embodiment, more than 70% of the cells are detained in the cell cycle. In another embodiment, at least 70% of the cells are detained in the cell cycle. In another embodiment, more than 75% of the cells are detained in the cell cycle. In another embodiment, at least 75% of the cells are detained in the cell cycle. In another embodiment, more than 82% of the cells are detained in the cell cycle. In another embodiment, at least 82% of the cells are detained in the cell cycle. In another embodiment, more than 85% of the cells are detained in the cell cycle. In another embodiment, at least 85% of the cells are detained in the cell cycle. In another embodiment, more than 87% of the cells are detained in the cell cycle. In another embodiment, at least 87% of the cells are detained in the cell cycle. In another embodiment, more than 90% of the cells are detained in the cell cycle. In another embodiment, at least 90% of the cells are detained in the cell cycle. In another embodiment, more than 92% of the cells are detained in the cell cycle. In another embodiment, at least 92% of the cells are detained in the cell cycle. In another embodiment, more than 93% of the cells are detained in the cell cycle. In another embodiment, at least 93% of the cells are detained in the cell cycle. In another embodiment, more than 94% of the cells are detained in the cell cycle. In another embodiment, at least 94% of the cells are detained in the cell cycle. In another embodiment, more than 95% of the cells are detained in the cell cycle. In another embodiment, at least 95% of the cells are detained in the cell cycle. In another embodiment, more than 96% of the cells are detained in the cell cycle. In another embodiment, at least 96% of the cells are stopped in the cell cycle. In another embodiment, more than 97% of the cells are detained in the cell cycle. In another embodiment, at least 97% of the cells are detained in the cell cycle. In another embodiment, more than 98% of the cells are detained in the cell cycle. In another embodiment, at least 98% of the cells are detained in the cell cycle. In another embodiment, more than 99% of the cells are detained in the cell cycle. In another embodiment, at least 99% of the cells are stopped in the cell cycle. Each possibility represents a different embodiment of the present invention.

In another embodiment, replication is reduced by 50% in relation to replication in a tissue culture plate. In another embodiment, replication is reduced by 60% in relation to a tissue culture plate. In another embodiment, replication is reduced by 65% in relation to a tissue culture plate. In another embodiment, the replication is

reduced by 70% in relation to a tissue culture plate. In another embodiment, replication is reduced by 75% in

relationship with a tissue culture plate. In another embodiment, replication is reduced by 80% in relation to a tissue culture plate. In another embodiment, replication is reduced by 85% in relation to a tissue culture plate. In another embodiment, replication is reduced by 90% in relation to a tissue culture plate. In another embodiment, replication is reduced by 95% in relation to a tissue culture plate. In another embodiment, the

Replication is reduced by 97% in relation to a tissue culture plate. In another embodiment, the replication is

reduced by 98% in relation to a tissue culture plate. In another embodiment, replication is reduced by 99% in

relationship with a tissue culture plate. Each possibility represents a different embodiment of the present invention.

In another embodiment, a method of the present invention further comprises the step of sowing later (for example, after culturing in the presence of a gel or a gel matrix of the methods of the present invention) the population of mesenchymal stem cells in a tissue culture apparatus. In another embodiment, the tissue culture apparatus contains induction medium. In another embodiment, the subsequent sowing stage is performed with chemical induction. Each possibility represents a different embodiment of the present invention.

This method also describes a method to study the proliferation or differentiation of a mesenchymal stem cell comprising the step of culturing the mesenchymal stem cell in a gel or a gel matrix with a stiffness in a range of 150-750 Pa, studying therefore the proliferation or differentiation of a mesenchymal stem cell.

The population of adipocytes of the methods of the present invention is, in another embodiment, a population comprising adipocytes. In another embodiment, the population is enriched with respect to adipocytes. In another embodiment, the population is a population of partially purified adipocytes. In another embodiment, the adipocytes are isolated from a biological source, followed by a stage of purification or enrichment. In another embodiment, the isolation of the biological source is done after cultivation. In another embodiment, the isolation of the biological source is done after cultivation and a stage of purification or enrichment. Each possibility represents a different embodiment of the present invention.

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In another embodiment, the cell population of the methods of the present invention is cultured in the presence of a gel or a gel matrix of the methods of the present invention. In another embodiment, the cell population is cultured in the gel or gel matrix. In another embodiment, the cell population is cultured in the gel or gel matrix. In another embodiment, the cell population is cultured in a tissue culture apparatus that contains the gel or gel matrix. Each possibility represents a different embodiment of the present invention.

In another embodiment "population of mesenchymal stem cells" refers to a population comprising mesenchymal stem cells (CMM). In another embodiment, the population is enriched with respect to CMM. In another embodiment, the population is a partially purified CMM population. In another embodiment, the CMMs are isolated from a biological source, followed by a stage of purification or enrichment. In another embodiment, the isolation of the biological source is done after cultivation. In another embodiment, the isolation of the biological source is done after cultivation and a stage of purification or enrichment. Each possibility represents a different embodiment of the present invention.

In another embodiment, the "mesenchymal" cells of the methods of the present invention are isolated or purified from bone marrow. In another embodiment, the cells are mesenchymal stem cells derived from bone marrow. In another embodiment, the cells are softened or purified from adipose tissue. In another embodiment, the cells are softened or purified from cartilage. In another embodiment, the cells are softened or purified from any other tissue known in the art. Each possibility represents a different embodiment of the present invention.

A gel or a gel matrix of the methods of the present invention have a shear modulus of 150-750 Pa.

In another embodiment, a gel or a gel matrix of the methods and compositions of the present invention have a stiffness equivalent to that of a biological tissue. In another embodiment, the biological tissue is bone marrow. In another embodiment, the biological tissue is fatty tissue. In another embodiment, the biological tissue is any other biological tissue known in the art. Each possibility represents a different embodiment of the present invention.

In one embodiment, the gel matrix described herein is capable of forming gels of various forces, depending on their structure and concentration as well as, in another embodiment, environmental factors such as ionic strength, pH and temperature. The combined viscosity and behavior of the gel called "viscoelasticity" in one embodiment, are examined by determining the effect that a swinging force has on the movement of the material. In another embodiment the elastic modulus (G '), viscous modulus (G ") and complex viscosity (n *) are the parameters to be changed using the methods described herein, and these are analyzed in another embodiment by modifying the tension or pressure harmoniously with time (Table 1). These parameters derive from the complex module (G *), which is the ratio of maximum tension to maximum pressure, and the phase angle (5), which is the angle at which the tension and pressure are out of phase.

Table ^ i = Relationship = enfr; e = modules = dynamic dynamic = deJ; aseJ6) = yjrecuenciaJw)

 Finished

 Symbol Definition Information provided

 Complex module

 G * [(G ') 2 + (G ”) 2] 0.5 All viscoelastic features

 Elastic module, storage module

 G 'G * cos 5 Energy stored for each deformation cycle; solid or elastic behavior

 Viscous module, loss module

 G ”G * sin 5 Energy dissipated per deformation cycle; liquid or viscous behavior

 Complex viscosity

 n * G * / o> Viscoelastic flow

In one embodiment, in the gel matrices described herein, part of the deformation caused by shear stress is elastic and will return to zero when the force is removed. The remaining deformation, such as the deformation created by the sliding displacement of the chains through the solvent in one embodiment, will not return to zero when the force is removed. Under a constant force, the elastic displacement remains constant in one embodiment, while the sliding displacement continues, increasing in this way.

In one embodiment, the term "elastic" or "elasticity," and similar terms refer to a physical property of the gel matrices described herein, namely the deformability of the gel under mechanical strength and the ability of the gel matrix. to retain its original shape when the deforming force is removed. In another embodiment, the term "elastic modulus" refers to Young's modulus and is a measure of the relationship of (a) the uniaxial tension along an axis of the material with respect to (b) the normal tension attached to it. along that axis.

The shear module (resulting from the changing tension) is the ratio of the shear stress to the shear pressure. From the complex relationship similar to the previous one it follows that:

image 1

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where G * is the complex shear module, G 'is the phase storage module, i is a material related factor and G ”is the loss module similarly directed out of phase; G * = E (G'2 + G ”2). The frequency with which these parameters intersect corresponds to a specific relaxation time (t) of the material.

In one embodiment, the linear viscoelastic properties of the gel matrices described herein are determined by measurements in an oscillating shear flow at small amplitude and with variable angular frequency. The values for G 'and G ”are largely determined here by the concentration of cellulose derivatives in the aqueous solution and the magnitude of the representative viscosity value. Therefore, hereinafter, only the relative evolution of G 'and G ”with increasing angular frequency (w) is considered. In another embodiment, at a concentration of 1.5 to 2% (w / w) of cellulose derivative of aqueous solution and at a temperature of approximately 20 ° C, the behavior of G 'and G "for cellulose derivatives is such that at a low angular frequency (w), the storage module G 'is smaller than the loss module G ", but with the increase in the angular frequency G' it increases more than G". In another embodiment, G ', above a certain angular frequency, finally becomes greater than G ”, and the solution at high angular frequency values therefore reacts predominantly elastically. This behavior is attenuated or changed using the modulation methods described herein.

In another embodiment, the term "elasticity" refers to the physical property of a material that defines its ability to deform by stress, whether reversible or not. As used herein, the elasticity and stiffness are inversely related and the elasticity (stiffness) of a material can be measured using an RFS III fluid spectrometer, available from Rheometrics, Piscataway, NJ, using a pressure of 2% oscillating shear at a frequency of 10 radians per second. The elasticity and other rheological properties of cells and other physiological tissues can be measured using any of a variety of methods known to those skilled in the art. Such methods may involve the use of reometers or atomic force microscopes, as an example (see, for example, Engler AJ, Rehfeldt F, Sen S, Discher DE, "Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation, "Methods Cell Biol. 2007; 83: 521-45; 15 Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA," Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion, "Cell Motil Cytoskeleton. Jan 2005; 60 (1): 24-34.).

In one embodiment, the expression "intrinsic viscosity" ([n *]) refers to the Kmite of the reduced viscosity extrapolated to zero concentration. Like the reduced viscosity, it has redproca concentration units, for example, ml g-1.

In one embodiment, stiffness or hardness refers to the G 'values observed or measured.

In another embodiment, a gel or a gel matrix of the methods of the present invention are coated with a solution comprising an adhesion protein. In another embodiment, the adhesion protein is a collagen. In another embodiment, the adhesion protein is a type I collagen. In another embodiment, the adhesion protein is a fibronectin. In another embodiment, the adhesion protein is any other adhesion protein known in the art. In another embodiment, the gel or gel matrix is coated with a solution comprising a combination of adhesion proteins. In another embodiment, the gel or gel matrix is coated with a solution comprising a collagen and a fibronectin. In another embodiment, the gel or gel matrix is coated with a solution comprising a type I collagen and a fibronectin. Each possibility represents a different embodiment of the present invention.

In another embodiment, the collagen of the methods of the present invention is a recombinant collagen. In another embodiment, the collagen is purified from a biological source. In another embodiment, the collagen is a type I collagen. In another embodiment, the collagen is any other type of collagen known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, the fibronectin of the methods of the present invention is a recombinant fibronectin. In another embodiment, the fibronectin is purified from a biological source. In another embodiment, fibronectin is a type I fibronectin. In another embodiment, fibronectin is any other type of fibronectin known in the art. Each possibility represents a different embodiment of the present invention.

The gelling agent of the methods of the present invention is, in another embodiment, an acrylamide. In another embodiment, the gelling agent is a mixture of acrylamide-bisacrylamide. In another embodiment, the gelling agent comprises acrylamide. In another embodiment, the gelling agent comprises a mixture of acrylamide-bisacrylamide. Each possibility represents a different embodiment of the present invention.

In another embodiment, an acrylamide gel of the methods of the present invention has an acrylamide: bisacrylamide ratio of between 100: 1 and 30: 1. In another embodiment, the acrylamide gel is prepared from a solution having an acrylamide: bisacrylamide ratio of between 100: 1 and 30: 1. In another embodiment, the ratio is between 100: 1 and 20: 1. In another embodiment, the ratio of acrylamide: bisacrylamide is between 100: 1 and 40: 1. In another embodiment, the ratio is between 100: 1 and 50: 1. In another embodiment, the ratio is between 100: 1 and 60: 1. In other

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realization, the ratio is between 100: 1 and 70: 1. In another embodiment, the ratio is between 120: 1 and 30: 1. In other

realization, the ratio is between 120: 1 and 40: 1. In another embodiment, the ratio is between 120: 1 and 50: 1. In other

realization, the ratio is between 120: 1 and 60: 1. In another embodiment, the ratio is between 120: 1 and 70: 1. In other

realization, the ratio is between 90: 1 and 20: 1. In another embodiment, the ratio is between 90: 1 and 30: 1. In another embodiment,

The ratio is between 90: 1 and 40: 1. In another embodiment, the ratio is between 90: 1 and 50: 1. In another embodiment, the ratio is between 90: 1 and 60: 1. In another embodiment, the ratio is between 80: 1 and 20: 1. In another embodiment, the ratio is between 80: 1 and 30: 1. In another embodiment, the ratio is between 80: 1 and 40: 1. In another embodiment, the ratio is between 80: 1 and 50: 1.

In another embodiment, the ratio is 30: 1. In another embodiment, the ratio is 20: 1. In another embodiment, the ratio is 25: 1. In another embodiment, the ratio is 35: 1. In another embodiment, the ratio is 40: 1. In another embodiment, the ratio is 45: 1. In another embodiment, the ratio is 50: 1. In another embodiment, the ratio is 55: 1. In another embodiment, the ratio is 60: 1. In another embodiment, the ratio is 65: 1. In another embodiment, the ratio is 70: 1. In another embodiment, the ratio is 75: 1. In another embodiment, the ratio is 80: 1. In another embodiment, the ratio is 85: 1. In another embodiment, the ratio is 90: 1. In another embodiment, the ratio is 95: 1. In another embodiment, the ratio is 100: 1.

Each acrylamide: bisacrylamide ratio represents a different embodiment of the present invention.

In another embodiment, an acrylamide gel of the methods of the present invention has a total acrylamide concentration of 3-5%. In another embodiment, the acrylamide gel is prepared from a solution that has a total acrylamide concentration of 3-5%. In another embodiment, the total acrylamide concentration is 2%. In another embodiment, the concentration is 2.5%. In another embodiment, the concentration is 3%. In another embodiment, the concentration is 3.5%. In another embodiment, the concentration is 4%. In another embodiment, the concentration is 4.5%. In another embodiment, the concentration is 5%. In another embodiment, the concentration is 5.5%. In another embodiment, the concentration is 6%. In another embodiment, the concentration is 2-5%. In another embodiment, the concentration is 2.5 to 5%. In another embodiment, the concentration is 3.5 to 5%. In another embodiment, the concentration is 2-4%. In another embodiment, the concentration is 2-4.5%. In another embodiment, the concentration is 2-5%. Each possibility represents a different embodiment of the present invention.

In another embodiment, the gelling agent of the methods of the present invention is a fibrin protein. In another embodiment, the gelling agent is a fibrinogen protein. In another embodiment, the fibrinogen is devoid of clotting factors. Each possibility represents a different embodiment of the present invention.

In another embodiment, the concentration of the recombinant fibrin or fibrinogen protein in a gel or a gel matrix of the methods of the present invention is 3-10 mg / ml. In another embodiment, the concentration is 3-12 mg / ml. In another embodiment, the concentration is 3-9 mg / ml. In another embodiment, the concentration is 3-8 mg / ml. In another embodiment, the concentration is 3-7 mg / ml. In another embodiment, the concentration is 3-6 mg / ml. In another embodiment, the concentration is 2-12 mg / ml. In another embodiment, the concentration is 2-10 mg / ml. In another embodiment, the concentration is 2-9 mg / ml. In another embodiment, the concentration is 2-8 mg / ml. In another embodiment, the concentration is 2-7 mg / ml. In another embodiment, the concentration is 2-6 mg / ml. In another embodiment, the concentration is 4-12 mg / ml. In another embodiment, the concentration is 4-10 mg / ml. In another embodiment, the concentration is 4-9 mg / ml. In another embodiment, the concentration is 4-8 mg / ml. In another embodiment, the concentration is 4-7 mg / ml. In another embodiment, the concentration is 5-12 mg / ml. In another embodiment, the concentration is 5-10 mg / ml. In another embodiment, the concentration is 5-9 mg / ml. In another embodiment, the concentration is 5-8 mg / ml. In another embodiment, the concentration is 2 mg / ml. In another embodiment, the concentration is 2.5 mg / ml. In another embodiment, the concentration is 3 mg / ml. In another embodiment, the concentration is 3.5 mg / ml. In another embodiment, the concentration is 4 mg / ml. In another embodiment, the concentration is 4.5 mg / ml. In another embodiment, the concentration is 5 mg / ml. In another embodiment, the concentration is 6 mg / ml. In another embodiment, the concentration is 7 mg / ml. In another embodiment, the concentration is 8 mg / ml. In another embodiment, the concentration is 9 mg / ml. In another embodiment, the concentration is 10 mg / ml. In another embodiment, the concentration is 11 mg / ml. In another embodiment, the concentration is 12 mg / ml. Each possibility represents a different embodiment of the present invention.

In another embodiment, a fibrin or fibrinogen protein of the methods of the present invention is a fibrin or fibrinogen protein of a heterothermal animal. In another embodiment, the fibrin or fibrinogen protein is a fibrin or fibrinogen protein of a homeotherm animal. In another embodiment, the fibrin or fibrinogen is from a fish. In another embodiment, the fibrin or fibrinogen is from a salmon. In another embodiment, the fibrin or fibrinogen is from any other fish known in the art. In another embodiment, the fibrin or fibrinogen is from any other heterotherm known in the art. In another embodiment, the fibrin or fibrinogen is from a mammalian. In another embodiment, the fibrin or fibrinogen is human fibrin or fibrinogen. In another embodiment, the fibrin or fibrinogen is fibrin or bovine fibrinogen. In another embodiment, the fibrin or fibrinogen is from any other mammal known in the art. In another embodiment, the fibrin or fibrinogen is from any other homeotherm known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, the gelling agent is agarose. In another embodiment, the gelling agent is agar. In another embodiment, the gelling agent is a glycosaminoglycan. In another embodiment, the gelling agent is a collagen. In another embodiment, the gelling agent is carrageenan. In another embodiment, the gelling agent is carrageenan. In another embodiment, the gelling agent is locust bean gum. In another embodiment, the gelling agent is glycerin. In

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Another embodiment, the gelling agent of the methods of the present invention is any other gelling agent known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, the gel or gel matrix of the methods of the present invention is a gel or a two-dimensional gel matrix. In another embodiment, the gel or gel matrix is a three-dimensional gel or gel matrix. In another embodiment, the gel or gel matrix is any other type of gel or gel matrix known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, a gel or a gel matrix of the methods of the present invention further comprises an animal serum. In another embodiment, the animal serum is a fetal bovine serum. In another embodiment, the animal serum is a bovine calf serum. In another embodiment, the animal serum is a horse serum. In another embodiment, the animal serum is any other type of animal serum that contains growth factor known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, a gel or a gel matrix of the methods of the present invention further comprises a protease inhibitor.

In another embodiment, a protease inhibitor of the methods of the present invention inhibits the function of a peptidase. In another embodiment, the protease inhibitor is a protein. In some embodiments, the protease inhibitor is a cystem protease inhibitor, a serine protease (serpine) inhibitor, a trypsin inhibitor, a threonine protease inhibitor, an aspartic protease inhibitor or a metalloprotease inhibitor. In another embodiment, a protease inhibitor is a suicide inhibitor, a transition state inhibitor or a chelating agent.

In another embodiment, the protease inhibitor is soy trypsin inhibitor (SBTI). In another embodiment, the protease inhibitor is AEBSF-HCl. In another embodiment, the inhibitor is (epsilon) -aminocaproic acid. In another embodiment, the inhibitor is (alpha) 1-anti-chymotrypsin. In another embodiment, the inhibitor is antithrombin III. In another embodiment, the inhibitor is (alpha) 1-antitrypsin ([alpha] 1-proteinase inhibitor). In another embodiment, the inhibitor is APMSF-HCl (4-amidinophenyl methane sulfonyl fluoride). In another embodiment, the inhibitor is sprotinin. In another embodiment, the inhibitor is benzamidine-HCl. In another embodiment, the inhibitor is chymostatin. In another embodiment, the inhibitor is DFP (diisopropylfluoro phosphate). In another embodiment, the inhibitor is leupeptin. In another embodiment, the inhibitor is PEFABLOC® SC (4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride). In another embodiment, the inhibitor is PMSF (phenylmethyl sulfonyl fluoride). In another embodiment, the inhibitor is TLCK (1-chloro-3-tosylamido-7-amino-2-heptanone HCl). In another embodiment, the inhibitor is TPCK (1-chloro-3-tosylamido-4-phenyl-2-butanone). In another embodiment, the inhibitor is trypsin inhibitor of egg white (ovomucoid). In another embodiment, the inhibitor is soy trypsin inhibitor. In another embodiment, the inhibitor is aprotinin. In another embodiment, the inhibitor is pentamidine isethionate. In another embodiment, the inhibitor is pepstatin. In another embodiment, the inhibitor is guanidinium. In another embodiment, the inhibitor is alpha2-macroglobulin. In another embodiment, the inhibitor is a zinc chelating agent. In another embodiment, the inhibitor is iodoacetate. In another embodiment, the inhibitor is zinc. Each possibility represents a different embodiment of the present invention.

In another embodiment, the amount of protease inhibitor that was used in the methods of the present invention is 0.1 mg / liter. In another embodiment, the amount of protease inhibitor is 0.2 mg / liter. In another embodiment, the amount is 0.3 mg / liter. In another embodiment, the amount is 0.4 mg / liter. In another embodiment, the amount is 0.6 mg / liter. In another embodiment, the amount is 0.8 mg / liter. In another embodiment, the amount is 1 mg / liter. In another embodiment, the amount is 1.5 mg / liter. In another embodiment, the amount is 2 mg / liter. In another embodiment, the amount is 2.5 mg / liter. In another embodiment, the amount is 3 mg / liter. In another embodiment, the amount is 5 mg / liter. In another embodiment, the amount is 7 mg / liter. In another embodiment, the amount is 10 mg / liter. In another embodiment, the amount is 12 mg / liter. In another embodiment, the amount is 15 mg / liter. In another embodiment, the amount is 20 mg / liter. In another embodiment, the amount is 30 mg / liter. In another embodiment, the amount is 50 mg / liter. In another embodiment, the amount is 70 mg / liter. In another embodiment, the amount is 100 mg / liter.

In another embodiment, the amount of protease inhibitor is 0.1-1 mg / liter. In another embodiment, the amount of protease inhibitor is 0.2-1 mg / liter. In another embodiment, the amount is 0.3-1 mg / liter. In another embodiment, the amount is 0.5-1 mg / liter. In another embodiment, the amount is 0.1-2 mg / liter. In another embodiment, the amount is 0.2-2 mg / liter. In another embodiment, the amount is 0.3-2 mg / liter. In another embodiment, the amount is 0.5-2 mg / liter. In another embodiment, the amount is 1-2 mg / liter. In another embodiment, the amount is 1-10 mg / liter. In another embodiment, the amount is 2-10 mg / liter. In another embodiment, the amount is 3-10 mg / liter. In another embodiment, the amount is 5-10 mg / liter. In another embodiment, the amount is 1-20 mg / liter. In another embodiment, the amount is 2-20 mg / liter. In another embodiment, the amount is 3-20 mg / liter. In another embodiment, the amount is 5-20 mg / liter. In another embodiment, the amount is 10-20 mg / liter. In another embodiment, the amount is 10-100 mg / liter. In another embodiment, the amount is 20-100 mg / liter. In another embodiment, the amount is 30-100 mg / liter. In another embodiment, the amount is 50-100 mg / liter. In another embodiment, the amount is 10-200 mg / liter. In another embodiment, the amount is 20-200 mg / liter. In another embodiment, the amount is 30-200 mg / liter. In another embodiment, the amount is 50-200 mg / liter. In another embodiment, the amount is 100-200 mg / liter.

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In another embodiment, the amount of protease inhibitor that is used in the methods of the present invention is 1000 u.i.k. (Kalicrema inactivating units) / liter. In another embodiment, the amount is 10 u.i.k./litro. In another embodiment, the amount is 12 u.i.k./litro. In another embodiment, the amount is 15 u.i.k./litro. In another embodiment, the amount is 20 u.i.k./litro. In another embodiment, the amount is 30 u.i.k./litro. In another embodiment, the amount is 40 u.i.k./litro. In another embodiment, the amount is 50 u.i.k./litro. In another embodiment, the amount is 70 u.i.k./litro. In another embodiment, the amount is 100 u.i.k./litro. In another embodiment, the amount is 150 u.i.k./litro. In another embodiment, the amount is 200 u.i.k./litro. In another embodiment, the amount is 300 u.i.k./litro. In another embodiment, the amount is 500 u.i.k./litro. In another embodiment, the amount is 700 u.i.k./litro. In another embodiment, the amount is 1500 u.i.k./litro. In another embodiment, the amount is 3000 u.i.k./litro. In another embodiment, the amount is 4000 u.i.k./litro. In another embodiment, the amount is 5000 u.i.k./litro

Each amount of the protease inhibitor represents a different embodiment of the present invention.

In another embodiment, the protease to which the protease inhibitor is directed from the methods of the present invention is a serious protease. In another embodiment, the protease is trypsin. In another embodiment, the protease is chymotrypsin. In another embodiment, the protease is carboxypeptidase. In another embodiment, the protease is aminopeptidase. In another embodiment, the protease is any other protease that acts in the duodenum or small intestine. Each possibility represents a different embodiment of the present invention.

The population of mesenchymal stem cells of the methods of the present invention is, in another embodiment, a population of adult mesenchymal stem cells. In another embodiment, the population of mesenchymal stem cells is a population of juvenile mesenchymal stem cells. In another embodiment, the population of mesenchymal stem cells is a population of infant mesenchymal stem cells. In another embodiment, the population of mesenchymal stem cells is a population of fetal mesenchymal stem cells. In another embodiment, the population of mesenchymal stem cells is a population of human mesenchymal stem cells. In another embodiment, the population of mesenchymal stem cells is of any animal known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, the cell type of interest is a stem cell. In another embodiment, the cell type of interest is a hematopoietic stem cell. In another embodiment, the cell type of interest is an adipocyte. In another embodiment, the cell type of interest is an endothelial progenitor cell. In another embodiment, the cell type of interest is a neural stem cell. In another embodiment, the cell type of interest is a progenitor cell resident in adult tissue. In another embodiment, the cell type of interest is a pancreatic progenitor cell resident in adult tissue. In another embodiment, the cell type of interest is a regenerative native beta cell. In another embodiment, the cell type of interest is a gastrointestinal stem cell. In another embodiment, the cell type of interest is a hepatopancreatic epithelial stem cell. In another embodiment, the cell type of interest is an epidermal stem cell. In another embodiment, the cell type of interest is an intestinal epithelial stem cell. In another embodiment, the cell type of interest is a retinal stem cell. In another embodiment, the cell type of interest is a neuronal epithelial stem cell. In another embodiment, the cell type of interest is a muscle stem cell. In another embodiment, the cell type of interest is an endothelial stem cell. In another embodiment, the cell type of interest is a peripheral blood stem cell. In another embodiment, the cell type of interest is any other type of stem cell known in the art.

In another embodiment, the cell type of interest is a progenitor cell. In another embodiment, the cell type of interest is a chondrogenic progenitor cell. In another embodiment, the cell type of interest is an adipogenic progenitor cell. In another embodiment, the cell type of interest is a marrow stromal progenitor cell. In another embodiment, the cell type of interest is a myogenic progenitor cell. In another embodiment, the cell type of interest is an osteogenic progenitor cell. In another embodiment, the cell type of interest is a tendon progenitor cell. In another embodiment, the cell type of interest is any other type of progenitor cell known in the art.

In another embodiment, the cell type of interest is a descending cell type. In another embodiment, the cell type of interest is a chondrocyte. In another embodiment, the cell type of interest is a stroma cell. In another embodiment, the cell type of interest is a myotube cell. In another embodiment, the cell type of interest is an osteocyte. In another embodiment, the cell type of interest is a tenocyte. In another embodiment, the cell type of interest is any other descendant cell type known in the art.

The method of the present invention comprises the step of incubating the mesenchymal stem cells in an induction medium. In another embodiment, the induction medium is a means of induction of stem cells. In another embodiment, the induction medium is an osteoblast induction medium. In another embodiment, the induction medium is a means of inducing hematopoietic stem cells. In another embodiment, the induction medium is an adipocyte induction medium. In another embodiment, the induction medium is a means of induction of endothelial progenitor cells. In another embodiment, the induction medium is a means of inducing neural stem cells. In another embodiment, the induction medium is a means of inducing progenitor cells resident in adult tissue. In another embodiment, the induction medium is a means of inducing pancreatic progenitor cells resident in adult tissue. In another embodiment, the induction medium is a means of

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Induction of regenerative native beta progenitor cells. In another embodiment, the induction medium is a means of induction of gastrointestinal stem cells. In another embodiment, the induction medium is a means of induction of hepatopancreatic epithelial stem cells. In another embodiment, the induction medium is an induction medium for epidermal stem cells. In another embodiment, the induction medium is a means of induction of intestinal epithelial stem cells. In another embodiment, the induction medium is a retinal stem cell induction medium. In another embodiment, the induction medium is a means of induction of neuronal epithelial stem cells. In another embodiment, the induction medium is a means of induction of muscle stem cells. In another embodiment, the induction medium is a means of induction of endothelial stem cells. In another embodiment, the induction medium is a means of induction of peripheral blood stem cells. In another embodiment, the induction medium is any other type of induction medium known in the art.

In another embodiment, the induction medium is a means of inducing progenitor cells. In another embodiment, the induction medium is a means of inducing chondrogenic progenitor cells. In another embodiment, the induction medium is a means of inducing adipogenic progenitor cells. In another embodiment, the induction medium is a means of induction of the marrow stromal progenitor cells. In another embodiment, the induction medium is an induction medium for myogenic progenitor cells. In another embodiment, the induction medium is an induction medium for osteogenic progenitor cells. In another embodiment, the induction medium is a means of induction of tendon progenitor cells. In another embodiment, the induction medium is any other type of progenitor cell known in the art.

In another embodiment, the induction medium is any other type of induction medium known in the art. Each possibility represents a different embodiment of the present invention.

The culture step of the methods of the present invention is carried out, in another embodiment, for at least 5 days. In another embodiment, the culture stage is performed for at least 4 days. In another embodiment, the cultivation stage is performed for at least 6 days. In another embodiment, the cultivation stage is performed for at least 7 days. In another embodiment, the cultivation stage is performed for at least 8 days. In another embodiment, the culture stage is performed for at least 10 days. In another embodiment, the culture stage is performed for at least 12 days. In another embodiment, the culture stage is performed for at least 15 days. In another embodiment, the cultivation stage is performed for at least 20 days. In another embodiment, the culture stage is performed for at least 25 days. In another embodiment, the cultivation stage is performed for at least 30 days. In another embodiment, the cultivation stage is performed for at least 35 days. In another embodiment, the cultivation stage is performed for at least 40 days. In another embodiment, the cultivation stage is performed for at least 50 days. In another embodiment, the cultivation stage is performed for at least 60 days. In another embodiment, the cultivation stage is carried out for more than 4 days. In another embodiment, the cultivation stage is carried out for more than 6 days. In another embodiment, the cultivation stage is carried out for more than 7 days. In another embodiment, the cultivation stage is carried out for more than 8 days. In another embodiment, the cultivation stage is performed for more than 10 days. In another embodiment, the cultivation stage is carried out for more than 12 days. In another embodiment, the cultivation stage is carried out for more than 15 days. In another embodiment, the cultivation stage is carried out for more than 20 days. In another embodiment, the cultivation stage is performed for more than 25 days. In another embodiment, the cultivation stage is performed for more than 30 days. In another embodiment, the cultivation stage is carried out for more than 35 days. In another embodiment, the cultivation stage is carried out for more than 40 days. In another embodiment, the cultivation stage is performed for more than 50 days. In another embodiment, the cultivation stage is carried out for more than 60 days. In another embodiment, the cultivation stage is performed for 4 days. In another embodiment, the cultivation stage is performed for 6 days. In another embodiment, the cultivation stage is performed for 7 days. In another embodiment, the cultivation stage is performed for 8 days. In another embodiment, the cultivation stage is performed for 10 days. In another embodiment, the cultivation stage is performed for 12 days. In another embodiment, the cultivation stage is performed for 15 days. In another embodiment, the cultivation stage is performed for 20 days. In another embodiment, the cultivation stage is performed for 25 days. In another embodiment, the cultivation stage is performed for 30 days. In another embodiment, the cultivation stage is performed for 35 days. In another embodiment, the cultivation stage is performed for 40 days. In another embodiment, the cultivation stage is performed for 50 days. In another embodiment, the cultivation stage is performed for 60 days. In another embodiment, the cultivation stage is carried out for more than 60 days. Each possibility represents a different embodiment of the present invention.

In another embodiment, the stage of maintaining the population of mesenchymal stem cells in a gel or a gel matrix of the methods of the present invention is preceded by a stage of culturing the mesenchymal stem cells in a tissue culture apparatus. In another embodiment, the tissue culture apparatus is a disk. In another embodiment, the tissue culture apparatus is a plate. In another embodiment, the tissue culture apparatus is a flask. In another embodiment, the tissue culture apparatus is a jar. In another embodiment, the tissue culture apparatus is a tube. In another embodiment, the tissue culture apparatus is any other type of tissue culture apparatus known in the art. In another embodiment, the culture stage is preceded by a culture stage of the mesenchymal stem cells in tissue culture medium; for example, not in the presence of a gel or a gel matrix of the methods of the present invention. In another embodiment, the stage of cell culture in a tissue culture apparatus or in tissue culture medium is performed after isolation of the mesenchymal stem cell population from a biological sample. In another embodiment, the culture stage is performed after purification of the population of mesenchymal stem cells from a biological sample. In another embodiment, the culture stage is performed after enrichment of the population of mesenchymal stem cells in a biological sample.

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Each possibility represents a different embodiment of the present invention.

In another embodiment, the culture step of the mesenchymal stem cell population in a gel or in a gel matrix of the methods of the present invention is performed directly after isolation of the mesenchymal stem cell population from a biological sample. In another embodiment, the culture stage is performed directly after the purification of the mesenchymal stem cell population from a biological sample. In another embodiment, the culture stage is performed directly after enrichment of the population of mesenchymal stem cells in a biological sample. "Directly" refers, in another embodiment, to a culture stage in the absence of first culture in a tissue culture apparatus. Each possibility represents a different embodiment of the present invention.

The population of progenitor cells of the methods of the present invention is, in another embodiment, a population of hematopoietic stem cells. In another embodiment, the population of progenitor cells is a precursor population of endothelial cells. In another embodiment, the population of progenitor cells is a population of satellite cells (for example, precursors of muscle cells). In another embodiment, the population of progenitor cells is a population of transit-amplifying neural progenitors of the rostral migratory stream. In another embodiment, the population of progenitor cells is a population of stromal cells in the bone marrow. In another embodiment, the population of progenitor cells is any other population of progenitor cells known in the art. Each possibility represents a different embodiment of the present invention.

"Population of progenitor cells" refers, in another embodiment, to a population comprising progenitor cells. In another embodiment, the population is enriched with respect to progenitor cells. In another embodiment, the population is a partially purified progenitor cell population. In another embodiment, the progenitor cells are isolated from a biological source, followed by a stage of purification or enrichment. In another embodiment, the isolation of the biological source is followed by cultivation. In another embodiment, the isolation of the biological source is followed by culture and a stage of purification or enrichment. Each possibility represents a different embodiment of the present invention.

Also described herein is a method for differentiating a transformed cell into a differentiated cell type, comprising the step of culturing the transformed cell in a gel or a gel matrix of the methods of the present invention, thus differentiating a cell transformed into a differentiated cell type. In another embodiment, the differentiated cell type is a progenitor cell. In another embodiment, the differentiated cell type is a descending cell type. In another embodiment, the differentiated cell type is a tissue cell type. In another embodiment, the differentiated cell type is one of the above cell types. In another embodiment, the differentiated cell type is any other type of differentiated cell type known in the art. Each possibility represents a different embodiment.

In another embodiment, a cell or cell population prepared by a method of the present invention is used to replace damaged tissues in a subject. In another embodiment, a cell or cell population prepared by a method of the present invention is used as a vehicle for antineoplastic agents (Kassem M, Ann N and Acad Sci. May 2006; 1067: 436-42). Each possibility represents a different embodiment of the present invention.

Methods for determining the proliferative capacity and differentiation force of mesenchymal stem cells are well known in the art, and are described, for example, in Baxter Ma et al (Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion Stem Cells. 2004; 22 (5): 675-82), Liu L et al. (Telomerase deficiency impairs differentiation of mesenchymal stem cells. Exp Cell Res. 10 Mar 2004; 294 (1): 1-8), and Bonab MM et al (Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 10 Mar 2006; 7: 14). Each possibility represents another embodiment of the present invention.

In another embodiment, an advantage of the methods of the present invention is the lack of immortalization of mesenchymal stem cells. In another embodiment, an advantage is the lack of evolution of cancer cells from a population of mesenchymal stem cells. In another embodiment an advantage is the preservation of the ability of the target cells to differentiate into multiple cell types. In another embodiment, an advantage is the preservation of the proliferative capacity of the cells. In another embodiment, one advantage is the preservation of the ability of the cells to support the growth of hematopoietic cells. In another embodiment, an advantage is the ability of the target cells to differentiate without a requirement of contact inhibition. In another embodiment, differentiation is a consequence of proliferation inhibition. Each possibility represents another embodiment of the present invention.

This document also describes methods to induce the proliferation of a quiescent somatic stem cell. It has been found that contacting a stem cell with a material that is less elastic than the elasticity of the naturally occurring in vivo microenvironment of the same type of stem cell is effective in inducing the proliferation of the stem cell. Embodiments of the disclosure therefore include contacting the somatic stem cell with a material comprising compounds that bind with integrins on the surface of the cell membrane and that have apparent elasticity for the stem cell less than the elasticity of the predominant material in the biological microenvironment of an in vivo somatic stem cell of the same type as the cell

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somatic mother For example, the proliferation of quiescent stem cells can be induced by placing them on a glass slide that has a stiffness of more than 1 gigaPascal (a small fraction of the elasticity of the mayonnaise of human or animal tissue types) and that is coated with a material that comes into contact with the integrins in the cell. In other embodiments, the proliferation of a stem cell can be induced by contacting the cell with an apparent cell material having an elasticity of less than 0.1 of the elasticity of the natural in vivo microenvironment of the stem cell. In other embodiments, proliferation can be induced by contacting the stem cell with a material that has an apparent elasticity for the stem cell of less than about 0.5 times (eg, about 0.4 to about 0.5 times) the elasticity of the natural in vivo microenvironment of the stem cell.

In embodiments, the cell may also be provided with nutrient growth material, for example, including growth and serum factors, to promote proliferation and maintain the biological activity of the stem cell and its offspring ex vivo. Those skilled in the art are aware of the formulation of the nutrient growth material for particular cell types and no variation of known nutrient growth materials for particular types of stem cells should be necessary for the practice of this aspect of the present invention.

Like other aspects of the present invention and the disclosure, embodiments of this proliferation inducing aspect with stem cells, including CMM, can be practiced. CMMs can be collected from living tissue, as described herein, or they can be obtained from other sources such as in vitro cultures and cryogenically frozen stem cells. Said cells may be in a quiescent state of natural origin or in an artificially induced quiescent state and may include, for example, cells in which quiescence has been induced or maintained using the methods of the present invention. Accordingly, the cells in which proliferation can be induced according to the methods described herein may include mesenchymal stem cells (CMM) derived from bone marrow, renal stem cells, liver stem cells, stem cells derived from skeletal muscle, bone-derived stem cells, CMM of dental pulp, CMM derived from cardiac muscle, CMM derived from synovial fluid, umbilical cord CMM and other types of cells that can be identified by one skilled in the art in light of the present specification.

The proliferation of stem cells according to the disclosure is reversible, allowing alternating states of quiescence and proliferation in a cell. For example, the methods described herein can be used to induce and maintain quiescence in a stem cell, for example, by contacting the stem cell with a material comprising compounds that bind with integrins on the cell surface and that have apparent elasticity for the cell approximately the same as the elasticity of the in vivo microenvironment of the cell. Then, proliferation in the cell can be induced by contacting the cell with a material comprising compounds that bind with integrins and have apparent elasticity for the cell substantially less than the elasticity of the in vivo microenvironment of the cell. Then, quiescence can be induced and maintained by contacting the resulting descending cells with a material that includes integrin binding compounds and having apparent elasticity for the cells approximately the same as the in vivo microenvironment of the cells.

In another embodiment, the proliferation of stem cells is induced in a quiescent state in accordance with a method described herein. Then, these quiescent cells can be induced to proliferate, as described above.

The present invention also provides methods for inducing the differentiation of a somatic stem cell in which biological activity is being maintained and quiescence has been induced or is being maintained in accordance with the methods of the present invention. Embodiments of this aspect of the present invention include the step of contacting said cells with a differentiation material comprising selected chemical stimuli to stimulate cell differentiation against a predetermined cell type and provide the cells with a nutrient material of differentiated cells to maintain the biological activity of the cells with stimulated differentiation. In some embodiments, the contacting stage may be preceded by a stage comprising inducing (or allowing) the proliferation, for example ex vivo, of the somatic stem cell by contacting the stem cell with a material that has a lower elasticity than the elasticity of the natural microenvironment of the intended differentiation target cell.

Differentiation can be induced in quiescent or proliferative stem cells maintained in biological activity according to the present invention, using methods known or evident to those skilled in the art in light of the present specification. For example, if the stem cell is a mesenchymal stem cell derived from human bone marrow, differentiation of the cell into adipocytes can be made by contacting the cell with an adipogenic medium (as analyzed in Example 1) on a substrate with an elasticity of approximately 250 Pa, as described in detail herein. Using information that can be obtained from this specification or as described in this specification, regarding the rheology of various types of tissues, as well as the means of differentiation to be used to induce stem cells to differ in different types of cells, it is apparent to those skilled in the art that the methods of the present invention can be used to induce a mesenchymal stem cell derived from bone marrow

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human is differentiated in one or more of at least the following types of cells: osteoblasts, chondrocytes, myocytes, adipocytes, pancreatic beta islet cells and neuronal cells. More generally, using information on rheology of various types of tissues and differentiation means used to induce differentiation of various types of stem cells in different types of cells, it will be readily apparent to those skilled in the art to induce differentiation in any of the types Stem cells identified herein, in one or more of an osteoblast, a chondrocyte, a myocyte, an adipocyte, a pancreatic beta islet cell, a neuronal cell or other type of cell.

In embodiments of this aspect of the invention, differentiated cells are contacted with a medium that includes nutrients to maintain the biological activity of the cells. For example, if a method of the present invention is used to induce a mesenchymal stem cell derived from human bone marrow to differentiate into adipocytes, the resulting adipocytes can be contacted with a nutrient medium to maintain their biological activity. The nutrient medium may comprise DMEM (glucose content balloon), fetal bovine serum and insulin. Other nutrient media, and methods for contacting differentiated cells with them, will be apparent to those skilled in the art in light of this specification.

This document also describes an artificial system to induce or maintain the quiescence and sustainable biological integrity of a somatic stem cell. In embodiments, the system includes an extracellular material ligand substance (MEC) for contacting a stem cell, a substrate material attached to the MEC ligand substance and a means for providing nutrients to the stem cell and maintaining its biological activity. . When the MeC ligand substance binds to the substrate material, it has elasticity similar to the elasticity of the predominant material in vivo in the biological microenvironment of an in vivo stem cell of the same type. Embodiments of the system can be adapted to induce quiescence in any of the cell types in which quiescence can be induced according to the methods of the invention described herein. For example, embodiments of the system can be adapted to induce quiescence in a somatic stem cell or an embryonic stem cell, a human stem cell or an animal stem cell, a somatic mesenchymal stem cell (CMM), a bone marrow derived CMM, a cell renal mother, a liver-derived stem cell, a CMM derived from skeletal muscle, a CMM derived from bone, a CMM from dental pulp, a CMM derived from cardiac muscle, a CMM derived from synovial fluid or a CMM from umbilical cord.

In embodiments, when the MEC ligand substance binds with the substrate and comes into contact with a stem cell, the MEC material binds with integrins on the surface of the stem cell so as to induce quiescence of the stem cell. , as described in detail herein.

In embodiments of said system, there may be a bonding material that links the MEC ligand material with the substrate material. For example, when the substrate material comprises a polyacrylamide gel and the MEC comprises a collagen-fibronectin mixture, the binding material can be NHS, specifically including N-hydroxysuccinimide acrylic acid ester. Depending on the nature of the substrate material and the extracellular material, those skilled in the art can easily determine bonding materials, which can also be characterized as crosslinking agents, to be used to bond the substrate material and the MEC in various embodiments of systems of the Present disclosure.

In embodiments, when the MEC ligand layer is coupled to the substrate, the MEC ligand layer has apparent elasticity for the stem cell substantially similar to the elasticity of the predominant material in the biological microenvironment of an in vivo stem cell of the same type as the stem cell that comes into contact with the ligand layer of MEC. This apparent elasticity of the MEC ligand layer may be independent of, almost independent of, or dependent on the elasticity of the substrate. In embodiments, the elasticity of the substrate is substantially similar to the elasticity of the predominant material in the biological microenvironment of an in vivo mother cell of the same type as the mother cell that comes into contact with the MEC ligand layer, and the ligand layer MEC presents the stem cell with substantially the same elasticity as the elasticity of the substrate with which it is coupled.

The artificial system described herein can be implemented using a variety of structures. In embodiments, the substrate material forms a matrix, and the MEC is dispersed in the matrix. In embodiments, a bonding material can also be dispersed in the substrate matrix to join the substrate matrix material with the MEC. For example, according to one embodiment, a 5: 1 mixture of collagen derived from rat tails (0.5 mg / ml) and fibronectin derived from humans (0.1 mg / ml), and an N- crosslinker Acrylic acid ester hydroxysuccinimide (NHS), are dispersed in a polyacrylamide gel. The gel is formulated so that the elasticity of the structure is approximately 250 Pa. When CMM derived from bone marrow are contacted with the polyacrylamide-NHS-fibronectin-collagen structure, it is induced to enter quiescence. When CMMs that come into contact with the structure are also provided with adequate nutrient material, their biological activity is maintained in a quiescent state.

In embodiments, the substrate material forms a layer, and the MEC forms a layer, directly or indirectly, bonded with the substrate layer. In other embodiments, the bonding material forms a bonding layer that joins the ligand layer

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of MEC with the substrate layer. The bonding layer can act to present the elasticity of the substrate to the MEC ligand layer so as to allow the MEC ligand layer to present that elasticity to the stem cell in which quiescence is to be induced. In one embodiment, the bonding layer includes appropriate crosslinking compositions and other materials that fulfill these functions. For example, the coupling layer may comprise NHS or, in specific embodiments, acrylic acid N-hydroxysuccinimide ester.

In embodiments, the substrate material is a polyacrylamide gel. As is known in the art, the elasticity of polyacrylamide gels can be adjusted by changing the acrylamide and bisacrylamide concentrations in the gel. It will be apparent to those skilled in the art, in the light of the present specification, how to prepare polyacrylamide gels or other gels or substrate materials for use in the systems of the present invention.

As is apparent further in the light of the present specification, the embodiments may use other structures. For example, an almost three-dimensional structure can be created by sowing stem cells in an MEC layer as described above, leaving the cells to rest on the MEC layer by submerging the system with cells in the middle, and placing another layer of MEC in the cells with Apparent apparent elasticity, as further described in Example 1.

In another embodiment, an advantage of the methods of the present invention is the resistance of the gel or gel matrix to proteolytic degradation. In another embodiment, an advantage is the resistance to active remodeling by the cells. In another embodiment, an advantage is the resistance of heterothermal fibrin (eg, salmon) to proteases secreted by marnffero neurons compared to mamffer fibrin (for example, human or bovine). In another embodiment, an advantage is the lower frequency of transfer of infectious disease of heterothermal fibrin (eg, salmon) compared to fibrin of mairnfero (for example human or bovine). Each possibility represents another embodiment of the present invention.

In another embodiment, the target cell of the methods of the present invention is an immortalized CMM.

This method also describes a method to induce the arrest of the growth of an immortalized CMM, which comprises the step of culturing the immortalized CMM in a gel or a gel matrix of the methods of the present invention, thereby inducing the growth arrest of an immortalized CMM. In another embodiment, a method is provided herein to inhibit the growth of an immortalized CMM population, which comprises the step of culturing immortalized CMM in a gel or a gel matrix of the methods of the present invention, inhibiting this way the growth of an immortalized CMM population. Each possibility represents a different embodiment.

In another embodiment, the target cell of the methods of the present invention is a transformed cell. In another embodiment, the transformed cell is a melanoma cell. In another embodiment, the transformed cell is any other type of transformed cell known in the art. Each possibility represents a different embodiment of the present invention.

As provided herein, M2 cells showed a larger size and larger cell populations in more nigged substrates. In another embodiment, an increased cell population is a result of increased cell growth in nigth substrates. In another embodiment, an increased population is due to the increased death of cells in soft substrates. Each possibility represents a different embodiment of the present invention.

This method also describes a method for inducing the arrest of the growth of a transformed cell, which comprises the step of culturing the transformed cell in a gel or a gel matrix of the methods of the present invention, thereby inducing the stopping the growth of a transformed cell. In another embodiment, a method is provided herein to inhibit the growth of a population of transformed cells, which comprises the step of culturing the transformed cell in a gel or a gel matrix of the methods of the present invention, inhibiting this way the growth of a population of transformed cells. Each possibility represents a different embodiment.

In another embodiment, a soft substrate of the methods of the present invention results in an increase in the inactive GDP-bound form of a GTP protein in the target cells. In another embodiment, the GTP protein is Rho. In another embodiment, the GTP protein is Rac. In another embodiment, the GTP protein is Cdc42. In another embodiment, the GTP protein is any other GTP protein known in the art. Each possibility represents a different embodiment of the present invention.

In another embodiment, the Rho family member signaled by forming focal adhesions. In another embodiment, Rho activation inhibits the expression of p2lWAF1 / c1p1. In another embodiment, p21 inhibition activates cyclin dependent kinases (CDK). In another embodiment, Rho activation induces the negative regulation and degradation of p27K1p1, another CDK inhibitor. In another embodiment, Rho activation induces ROCK, resulting in the activation of the Ras-Raf-MEK-ERK route. In another embodiment, this route induces the transcription of cyclin D1 mediated by Ras. In another embodiment, this induces the progression of the G1 phase. Overall, it was shown that the

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Rho activation led to progression of the G1 phase. In another embodiment, the activation of Rac or Cdc42 positively regulates cyclin E1 and cyclin D1, resulting in the progression of the G1 phase. In another embodiment, the activation of a small GTP binding protein of the Rho family results in signal transduction to enhance cell cycle progression. Each possibility represents a different embodiment of the present invention.

In another embodiment, a soft substrate of the methods of the present invention reduces the contractility of the actomyosin system of the target cell. In another embodiment, this induces the target cells to cease proliferation. In another embodiment, this induces the target cells to become competent for additional stimuli to restart proliferation. In another embodiment, this induces the target cells to be competent for additional stimuli to commit to terminal differentiation. Each possibility represents a different embodiment of the present invention.

In another embodiment, a soft substrate of the methods of the present invention induces actomyosin activation. In another embodiment, the soft substrate induces Rho-regulated actomyosin. In another embodiment, the soft substrate induces myosin II. In another embodiment, the soft substrate induces myosin II regulated by Rho. Each possibility represents a different embodiment of the present invention.

In another embodiment, a composition of the methods of the present invention further comprises an activator of a member of the Rho family. In another embodiment, the composition further comprises an inhibitor of a member of the Rho family. In another embodiment, the Rho family member is Rho. In another embodiment, the Rho family member is Rac. In another embodiment, the Rho family member is Cdc42. In another embodiment, the member of the Rho family is any other member of the Rho family known in the art. Each possibility represents a different embodiment of the present invention. In another embodiment, the composition further comprises an actomyosin activator. In another embodiment, the composition further comprises an actomyosin inhibitor. In another embodiment, the composition further comprises a myosin II activator. In another embodiment, the composition further comprises a myosin II inhibitor. Each possibility represents a different embodiment of the present invention.

Methods for polymerizing acrylamide gels are well known in the art. In another embodiment, 1.5 pl of TEMED (FisherBiotech CAS # 110189) and 5 pl of 10% ammonium persulfate are added with the appropriate amount of H2O to produce a final volume of 1,000 pl, the solution is transferred with a Pipette to a coverslip and on top of the solution a top coverslip is placed, then, 10 minutes later, it comes off. Each method represents a different embodiment of the present invention.

In another embodiment, a heterobifunctional crosslinker is used to crosslink adhesion proteins in the gel or gel matrix. In another embodiment, the heterobifunctional crosslinking agent is sulfo-SANPAH (sulfosuccinimidyl6 (4'-azido-2'-nitrophenyl-amino) hexanoate, Pierce No. 22589). In another embodiment, the heterobifunctional crosslinker is any other heterobifunctional crosslinker known in the art. In another embodiment, sulfo-SANPAH is used as follows. Sulfo-SANPAH 1 mg / ml is dissolved in H2O, and with a pipette, 200 µl of this solution is transferred to the gel surface. The polyacrylamide gel is then placed under an ultraviolet lamp at a distance of 15.24 cm and irradiated for 10 min. Then, it is washed three times, using, in each wash, 3 ml of 200 mM HEPES, pH 8.6. After the last HEPES solution is aspirated, 200 µl of a 0.14 mg / ml fish fibronectin solution (Sea Run Holdings, South Freeport, ME) or type I collagen 0.14 mg / is transferred with a pipette ml on the polyacrylamide gel. The multiwell plate that houses the gels is then incubated at 5 ° C for 4 h. Each method represents a different embodiment of the present invention.

To assemble a system as described above or to otherwise carry out the methods of the present invention, a kit of components may be provided. In one embodiment, said kit includes as distinct components: (1) a substrate, or materials for preparing a substrate; (2) optionally, materials for preparing and / or applying a coating layer; and (3) materials for preparing and / or applying MEC ligand material to form a MEC ligand layer. The substrate component may, for example, comprise a polyacrylamide gel of a predetermined elasticity, a polyacrylamide gel with materials to adjust the elasticity of the gel to a predetermined elasticity or range of elasticities, or materials to prepare a gel or other substrate suitable with the desired elasticity. In embodiments, the kit includes a device for mounting or holding the gel to facilitate application of the MEC ligand layer and optionally a coupling layer to couple the substrate to the MEC ligand layer. As is apparent to those skilled in the art in light of the present specification, to facilitate the storage, shipping and assembly of the kits, various components of the kits described herein can be combined.

Embodiments of said kit may also include materials for preparing and / or applying the MEC ligand layer. For example, the kit may include MEC ligand material for direct application to the substrate. In other embodiments, the kit may include individual components or ingredients of the MEC ligand layer material, with instructions for preparing it and applying it to the other components of the kit. Optionally, the kit may include materials and instructions for assembling and applying a coupling layer to couple the substrate to the

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MEC ligand layer. In a specific embodiment, a kit of a system described herein to induce quiescence in a CMM derived from human bone marrow includes the following:

(a) a glass Petri dish (or other apparatus for supporting the substrate and containing the rest of the components);

(b) materials for preparing a polyacrylamide gel with a hardness of approximately 250 Pa to act as the substrate, including appropriate amounts of acrylamide and bisacrylamide;

(c) Acrylic acid N-hydroxysuccinimide (NHS) ester crosslinker to apply to the gel to form a coupling layer; Y

(d) materials for preparing a ligand layer of MEC, including the materials identified in Example 1, to prepare a 1: 5 fibronectin-collagen extracellular material.

In one embodiment, a kit of the present disclosure comprises a polyacrylamide gel formulated with an elasticity to induce quiescence in stem cells of a specific type; a solution that includes crosslinking compositions for application to the gel; extracellular material formulated to bind with integrins on the surface of the specified cell type; and, optionally, suitable nutrient material (which users can also prepare) to induce quiescence in the cells.

In some embodiments of the kit, the kit comprises a layer of material that provides both elasticity and MEC ligands. In another embodiment, the kit may comprise the following three layers of materials:

a) a gel substrate that confers elasticity to the system;

b) MEC ligands; Y

c) crosslinkers that connect MEC ligands with the substrate.

In kit embodiments of the present disclosure, the kit components are placed in a receptacle, such as a plastic bag or container containing phosphate buffered saline (PBS). In some embodiments, the receptacle may also contain preservatives such as sodium azide. The components can be hydrated in the kit, for example, during storage. In some embodiments, the receptacle is sealed and / or protected with appropriate frames to prevent damage to the system. The kit can be shipped at a low temperature, such as at a temperature of about 2 ° C to about 8 ° C.

In kit embodiments of the present disclosure, users can open the receptacle and place the kit components in an appropriate material, such as a tissue culture plate. Users can also remove any hard material on the components to expose the MEC ligands and wash the components with a buffer such as PBS. Users can then plant cells in the system by putting a cell suspension comprising cells, medium and serum, as necessary.

The methods of the present invention can be practiced in vivo as well as ex vivo. Such systems may include, for example, porous structures for insertion in specific tissues or in the circulatory system to maintain a quiescent stem cell in the organism. For example, corresponding stem cells, MECs and, optionally, binding material, can be dispersed in a polymeric matrix that has apparent elasticity apparent for the stem cells to induce or maintain quiescence, and which also has sufficient porosity to allow nutrients to enter. alive reach the cell and allow the proteas and other factors expressed by the cell to leave the matrix. Other embodiments may include cassettes or other devices that induce or maintain quiescence of the stem cells, and that may be implanted in a host.

This document also describes a quiescent stem cell maintained in biological activity ex vivo. Examples of such a stem cell include a somatic stem cell or an embryonic stem cell, a human stem cell or an animal stem cell, a mesenchymal stem cell (CMM), CMM derived from bone marrow, a renal stem cell, a derived stem cell Hepatica, a CMM derived from skeletal muscle, a CMM derived from bone, a CMM from dental pulp, a CMM derived from cardiac muscle, a CMM derived from synovial fluid or a CMM from umbilical cord. In embodiments, the methods of the present invention are used to induce or maintain a stem cell in a quiescent state, and to maintain the biological activity of said quiescent stem cells.

Accordingly, an apparatus for modulating the growth of a mesenchymal stem cell is also described herein, comprising: a gel matrix having a stiffness in the range of 150-750 Pa; and an adipocyte induction medium, wherein said gel or said gel matrix is coated with a type I collagen, with a fibronectin or with a combination thereof.

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In other embodiments, gels and gel matrices of any of the methods described above have any of the characteristics of a gel or a gel matrix of the methods of the present invention. Each feature represents a different embodiment of the present invention.

Experimental details section

Example 1: Measurement of stiffness of various tissues and preparation of polyacrylamide gels that approximate tissue stiffness

Materials and experimental methods Preparation of polyacrylamide gels

Acrylamide and bisacrylamide solutions (Fisher Biotech, Loughborough, Leicestershire, UK) were prepared containing a constant polymer mass of 7.5% and bisacrylamide concentrations of 0.01%, 0.03% or 0.3% to alter the rigidity. Acrylamide, bisacrylamide, ammonium persulfate and N, N, N ', N'-Tetramethylenediamine (TEMED) under a non-aqueous layer of toluene containing 0.5% acrylic acid N-hydroxysuccinimide ester (Sigma, St. Louis, MS) was polymerized between two coverslips, chemically modified as follows: 200 pl of 0.1 N NaOH were transferred by pipette to cover the surface of a 25 mm diameter glass coverslip (Fisherbrand, catalog # 12-545-102; Fisher Scientific, Pittsburgh, PA)) for 5 minutes. The NaOH solution was aspirated, and 200 pl of 3-APTMS (3-aminopropyltrimethoxylan, Sigma No. 28-1778, St. Louis, MO) was applied for 3 min. The glass slide was carefully rinsed with deionized water to wash off any remaining 3-APTMS solution and 200 µl of 0.5% v glutaraldehyde (Sigma No. G7651) in H2O was added over the slide for 20 minutes. The glass coverslip was rinsed with water, an 18 mm diameter glass coverslip was placed on top of a piece of parafilm inside a tissue culture plate, and a few drops of a 10-fold Surfasil solution were transferred by pipette Volume% (Pierce No. 42800, Pierce, Rockford, IL) in chloroform on the parafilm near the coverslip. The tissue culture plate with a half closed lid was placed in a vacuum evaporator for 10 minutes.

The N-succinimidyl acrylate incorporated into the gel surface was reacted with 0.2mg / ml of laminin (Collaborative Biomedical, Bedford, MA) to produce a uniform coating of adhesive ligands. After washing with HEPES buffer to remove traces of non-polymerized solvent, wells containing polyacrylamide (PA) gels were loaded with culture medium and allowed to equilibrate overnight at 35 ° C.

Viscoelastic characterization of material frames

The dynamic shear modules of the gels were measured in a tension-controlled, rheometric fluid III spectrometer (Rheometrics, Piscataway, NJ). A sample of 500 pl was polymerized between two steel plates, and the shear modulus G '(w), which describes the elastic resistance, was calculated from the shear stress in phase with an oscillating shear stress of 2% (1 rad / s). The dynamic shear modules of the tissues were measured in a similar manner. An 8 mm diameter sample was cut using a stainless steel punch and placed between the plates. The prolonged G '(u>) was measured by oscillation at a tension of 2% and the prolonged shear modulus G (t) was measured by applying a continuous tension at 10% and allowing the sample to stand for 30 seconds.

RESULTS

Polyacrylamide gels were prepared and coated with a mixture of 0.14 mg / ml type I collagen and 0.14 mg / ml fish fibronectin. By adjusting the acrylamide and bisacrylamide concentration, a wide range of stiffness was achieved (Figure 1A). The stiffness of polyacrylamide gels does not affect the amount or distribution of the extracellular matrix in the gels. In vitro reometna was also used to determine the elastic properties of tissues relevant to mesenchymal stem cells (Table 1). Polyacrylamide gels with G 'of 200 Pa were prepared (used herein as an example of "soft gels" and referred to as such) or 7,500 Pa (used herein as an example of "nid gels" and referred to as such); Soft gels mimic the stiffness of fatty tissues and bone marrow.

Table 1. Tissue stiffness. Rat tissues from three Sprague-Dawlev rats were obtained.

 Tissue

 Rigidity (Pa)

 Bovine bone marrow

 225 ± 25

 Subcutaneous rat fat

 157 ± 36

 Rat visceral fat

 130 ± 40

 Rat liver

 403 ± 28

 Skeleton Rat Muscle

 2251 ± 166

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Therefore, polyacrylamide gels were prepared that mimicked the stiffness of biological tissues. Example 2: Cellular form and structure of CMMh F-actin in soft and nidged gels MATERIALS AND EXPERIMENTAL METHODS

CMMh were seeded sparingly in neat gels or soft gels coated with type I collagen and fibronectin. The cells were incubated for 24 hours in DMEM + 10% fetal calf serum, fixed and stained with Alexa Fluor 488 phalloidin.

RESULTS

The effect of the rigidity of the extracellular matrix on the form and structure of CMMh F-actin (human mesenchymal stem cells) was investigated. The cells were incubated in the presence of serum in matrices with various stiffnesses for 24 hours to allow adhesion and propagation. The CMMh seeded in rigid gels or glass took a tapered shape and showed tension fibers and cortical F-actin (as shown by staining with Alexa Fluor 488 phalloidin), while the cells seeded in soft gels showed a rounded appearance, lacking Tension fibers and contain F-actin aggregates (Figure 1B-C).

Therefore, the CMMh detect the rigidity of the extracellular matrix, which influences its form and structure of F-actin.

Example 3: Inhibition of the proliferation of CMMh in soft gels.

EXPERIMENTAL MATERIALS AND METHODS

CMMh were incubated with BrdU (Invitrogen, Carlsbad, CA) overnight in the presence of serum. The cells were fixed and immunostained for BrdU (Invitrogen). More than 50 cells were counted three times in randomly chosen fields.

RESULTS

The incorporation of 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdU) in CMMh was measured as a marker of cell cycle progression (Figure 2). Cells were seeded dispersed in soft gels, nigid gels or on glass surfaces, all of which were coated with type I collagen and fibronectin. As a control, confluent cells were also prepared on glass surfaces. As expected, CMMh seeded dispersed on glass surfaces effectively incorporated BrdU, indicating a high level of proliferation. When the cells were confluent on the glass surfaces, very few CMMh incorporated BrdU due to contact inhibition. 42% of the CMMh in rigid gels incorporated BrdU, indicating that a large population of cells was proliferating, although significantly less than that of cells seeded dispersed on a glass surface. On the other hand, no CMMh in soft gels incorporated BrdU, even though the cells were viable as assessed by the absence of trypan blue staining. In addition, the continuous incubation of CMMh seeded in dispersed form in matrices produced a different cell density depending on the stiffness of the matrices with higher density in sharper matrices.

Therefore, soft matrices inhibit the proliferation of CMMh even in the presence of serum.

Example 4: CMMh in soft gels are competent to differentiate into adipocytes

EXPERIMENTAL MATERIALS AND METHODS

Adipocyte differentiation studies

CMMh of cell suspensions were seeded in 96-well tissue culture plates coated with type I collagen plus fibronectin at a density of 3.5 x 104 cells / well. After incubating the cells in DMEM containing 10% FCS (culture medium, "MC") for 24 hours, the differentiation of the cells into adipocytes was induced by incubating for 3 days (2 cell cycles) in adipogenic induction medium (MIA ) (MC, 1 pM dexamethasone, 200 pM indomethacin, 10 pg / ml insulin and 0.5 mM methyl isobutylxanthine) then maintained in adipogenic maintenance medium (MC, 10 pg / ml insulin). 8 days after switching to MIA, adipocyte differentiation was assessed either by staining with Oil Red O (Sigma-Aldrich, St. Louis, MO) or by immunostaining with respect to PPARy2 using antibodies against PPARy2. More than 50 cells were counted three times in randomly chosen fields. The antibodies against PPARy2 were provided by Dr. Mitchell A. Lazar, University of Pennsylvania.

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RESULTS

To confirm the viability of CMMh in soft gels, their ability to differentiate into adipocytes was measured in two ways; (a) immunostaining of receptors activated by peroxisome gamma 2 proliferators (PPARy2), one of the key transcription factors for adipogenesis, and (b) staining with Oil Red O to measure the accumulation of Kpidos. When differentiation in confluent CMMh adipocytes on a glass surface was induced by a mixture of dexamethasone, indomethacin, 3-isobutyl-1-methyl-xanthine and insulin in medium containing fetal calf serum, approximately 40% of the cells showed an adipocyte phenotype (Figure 3A). On the contrary, in soft gels with induction, the differentiation rate reached more than 80%, significantly higher than that of glass; without induction, no differentiation of adipocytes was observed. In addition, the CMMh seeded dispersed in glass showed a high level of proliferation (Figure 2) and did not differentiate into adipocytes (Figure 3B). These results also indicate that CMMh may have to leave the cell cycle as a prerequisite for terminal differentiation.

Therefore, CMMh seeded dispersed in soft gels are completely viable and are competent for adipocyte differentiation.

Example 5: Structure of F-actin in astrocytes seeded in nigid or soft gels

To further characterize and quantify the response of the cells to the stiffness of the matrix, the effect of the stiffness of the extracellular matrix on the structure of F-actin in astrocytes was also tested. Primary astrocytes were isolated from embryos of Sprague-Dawley rats as follows: embryos (E17-E19) were removed by caesarean section of a Sprague-Dawley rat with programmed gestation and the cortices were removed. The tissue was digested with trypsin / DNase at 37 ° C, centrifuged (1000 g x 5 min), and filtered to give a cell suspension. For cultures containing both neurons and glial cells, the cells were seeded directly on plates with substrates. Primary astrocyte cultures were maintained for 14 days in culture with a series of trypsinizations to remove neurons. The cultures used for the experiments were> 98% astrocytes as determined by GFAP immunocytochemistry. The cells were grown in a 37 ° C incubator and with 5% CO2 in Dulbecco's modified Eagle's medium (BioWhittaker, East Rutherford, NJ) supplemented with Ham's F12 (Sigma) and 5% fetal bovine serum (Hyclone, Logan , UT) for 7 days followed by 5 additional days of culture in Neurobasal (Gibco, Carlsbad, CA) also supplemented with 5% fetal bovine serum, 2 mM 1-glutamine, 50 mcg / ml streptomycin and 50 units / ml penicillin.

The cells were incubated in the presence of serum in nigged (11 kPa) or soft (150 Pa) polyacrylamide gels for 48 hours to allow adhesion and propagation. The cells were fixed, and the structure of F-actin with phalloidin was visualized. As shown in Figure 4, tension fibers and cortical F-actin were observed in astrocytes seeded in nested gels. On the contrary, in astrocytes seeded in soft gels, there were no tension fibers and only cortical actin carcasses were observed. Therefore, astrocytes seeded in soft gels detected the flexibility of the matrix and consequently showed no tension fibers.

Example 6: Low level of RHO GTP load in astrocytes seeded in soft gels

EXPERIMENTAL MATERIALS AND METHODS

Rhotequina Extraction Test

The cells were washed with ice cold Tris buffered saline and lysed in RIPA buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 NaCl mM, 10 mM MgCh, 10 pg / ml each of leupeptin and aprotinin, and 1 mM PMSF). Cell lysates were clarified by centrifugation at 13,000 x g at 4 ° C for 10 min, and equal volumes of lysates were incubated with GST-RBD beads (20 pg) at 4 ° C for 45 min. The beads were washed 4 times with buffer B (Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCh, 10 mg / ml each of leupeptin and aprotinin and 0.1 mM PMSF). Bound Rho proteins were detected by Western blotting using a monoclonal antibody against RhoA (Santa Cruz Biotechnology). Densitometric analysis was performed using Alphalmager ™ system (Alpha Innotech). The amount of Rho bound to RBD was normalized with respect to the total amount of Rho in cell lysates for comparison of Rho activity (Rho level bound to GTP) in different samples.

RESULTS

To determine whether the loss of tension fibers induced by soft gels in astrocytes is associated or not with Rho inactivation, astrocytes were seeded in polyacrylamide gels with various stiffnesses and incubated in the presence of serum for 48 hours. Cell lysates were prepared and Rho gTp loading was assayed using GST-rhotequina and a rhotequina extraction assay. The ratio of Rho bound to GTP was calculated against the total. Astrocytes seeded in soft gels showed a low level of Rho bound to GTP (Figure 5), indicating the attenuation of Rho activity in astrocytes seeded in a soft matrix, resulting in the absence of tension fibers in the cells

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Example 7: Melanoma cells modulate their spread. based on the rigidity in the extracellular matrix MATERIALS AND EXPERIMENTAL METHODS

M2 cells were seeded sparingly in matrices of various rigidities coated with a mixture of type I collagen and fibronectin. After 24 hours of incubation. The cell area was measured by delimiting the cellular Kmites. More than 30 cells were counted 3 times in randomly chosen fields.

RESULTS

To test whether the transformed cells modulate their behavior according to the level of stiffness in the extracellular matrix. The effect of stiffness on cell propagation in human melanoma cell lines, called M2 cells, was measured. As shown in Figure 6. M2 cells showed a larger size in more nigged substrates. Therefore, the transformed M2 cells have a capacity to modulate their behavior (for example, cell propagation) according to the mechanical properties of the matrix.

Example 8: Reduced amount of melanoma cells in soft gels

To determine the effect of the stiffness of the matrix on the size of the population of M2 cells. M2 cells were seeded in soft or nested gels (the same number in each) and coated with a mixture of type I collagen and fibronectin. The effectiveness of cell adhesion to each substrate was evaluated after 24 hours of incubation in the presence of serum. when M2 cells adhered completely and proliferated in both gels. As shown in Figure 7. there were no significant differences in the number of adhered cells between soft and neat gels. which indicates that the stiffness of the matrix does not affect the adhesion of M2 cells. After an incubation of 72 hours. although the same number of cells adhered to each substrate (Figure 7). the additional 48 hours of incubation caused a significantly larger population of cells in nigged gels (Figure 8). No observable difference was observed in the number of cells that floated in the middle between soft and nested gels after 72 hours of incubation.

These results also demonstrate methods for quantifying CMMh responses to soft substrates.

Example 9: Use of soft gels for prolonged conservation of CMMh without attenuating viability and self-renewal

EXPERIMENTAL MATERIALS AND METHODS

Soft polyacrylamide gels with G 'of approximately 200 Pa coated with a mixture of type I collagen and fibronectin in glass coverslips are prepared as described in Example 1. Polyacrylamide gels are placed in gel-coated 6-well plates of 1% agarose. to prevent cell adhesion outside polyacrylamide gels. or glass coverslips. 5 x 104 CMMh of pass 2 are seeded in soft polyacrylamide gels or directly in 6-well tissue culture plates coated with type I collagen plus fibronectin. and incubated in DMEM supplemented with 10% fetal calf serum (FCS). Identical CMMh samples are suspended in DMEM containing 10% FCS and 10% dimethylsulfoxide (dMsO) and kept frozen in liquid nitrogen vapor according to the conventional protocol (Gordon SL et al. Cryobiology. Sep 2001; 43 (2) : 182-7). After reaching 90% confluence. cells that had been seeded directly in tissue culture plates are trypsinized and subcultured in new 6-well tissue culture plates at a density of 5 x 104 cells / well. Cells in tissue culture plates are maintained until they reach pass 10. Cells seeded in soft polyacrylamide gels are fed again with new DMEm supplemented with 10% FCS twice every 7 days. When the CMMh maintained in tissue culture plates reach pass 10. CMMh reserved in liquid nitrogen vapor is thawed to generate a cell suspension.

RESULTS

To determine the viability of CMMh subjected to prolonged preservation in a quiescent state in soft gels. CMMh are stored in soft gels until the CMMh maintained in tissue culture plates reach pass 10. The viability of these cells is compared with that of the reserved cells in liquid nitrogen vapor. Cells attached to soft gels or tissue culture plates are trypsinized. while the cells stored in liquid nitrogen are thawed. to generate a cell suspension. Viability is determined by trypan blue staining of cell suspensions. Thus. Soft gel storage is an effective means of maintaining the viability of CMMh.

To measure the proliferation force of the CMMh stored in prolonged incubation in soft gels. cells that have been kept in soft gels are prepared. in tissue culture plates. or that have been kept frozen in liquid nitrogen vapor. and a BrdU incorporation test is performed by re-seeding cells from each source in tissue culture plates coated with type I collagen plus fibronectin and incubating

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in the presence of serum and BrdU for 12 hours. The cells are fixed and immunostained with respect to BrdU by incubating cells with anti-BrdU antibodies (Invitrogen).

Example 10: Use of soft gels for prolonged conservation of CMMh without attenuating differentiation MATERIALS AND EXPERIMENTAL METHODS

Osteoblast differentiation assays

Pass-through CMMh suspensions 10 are seeded in 96-well tissue culture plates coated with type I collagen plus fibronectin at a density of 103 cells / well. After incubating the cells in MC for 24 hours, the differentiation of the cells into osteoblasts is induced by changing the medium to osteogenic induction medium (MIO) (MC, 50 jM ascorbic acid-2-phosphate, 10 mM p-glycerophosphate, and 100 nM dexamethasone), changing the medium every 3 days for 3 weeks. Osteoblast differentiation is evaluated by fixing the cells with acetone / citrate and tining with respect to alkaline phosphatase activity with Fast Blue RR / naphthol (Sigma-Aldorich, Kit No. 85).

RESULTS

Next, the strength of differentiation of CMMh subjected to prolonged preservation in a quiescent state in soft gels is measured. CMMh stored in soft gels or in tissue culture plates are trypsinized to make generated suspensions; in parallel, cell suspensions are prepared by thawing the CMMh kept frozen in liquid nitrogen. Adipocyte differentiation is evaluated as described in Example 4.

In additional studies, the production of adipocytes is measured after incubating CMMh directly in the soft gel, without previously sowing in tissue culture plates and trypsinization.

In additional studies, the production of osteoclasts in cell suspensions prepared from soft gels, tissue culture plates or frozen storage is measured.

In additional studies, the production of osteoclasts is measured after incubating CMMh directly in the soft gel, without previously sowing in tissue culture plates and trypsinization.

Example 11: Involvement of small GTP binding proteins of the Rho family and actomyosin system in the regulation of stem cell growth by matrix stiffness

EXPERIMENTAL MATERIALS AND METHODS

Rho family trials

Polyacrylamide gels with G 'of approximately 200 Pa (soft gels) and with G' of approximately 7500 Pa (nested gels) are prepared in glass coverslips, and gels and coverslips are coated with a mixture of type I collagen and fibronectin. Polyacrylamide gels or glass coverslips are placed in 6-well plates covered with 1% agarose gel to prevent cell adhesion outside polyacrylamide gels or glass coverslips. 5 x 104 CMMh are seeded in a polyacrylamide gel or glass coverslip, then incubated in the presence of serum for 24 hours. The cells are subjected to a precipitation test to investigate the GTP load level of Rho, Rac and Cdc42 using their activation assay kit (Upstate Biotech, Charlottesville, VA).

Transfection of cells with dominant or negative constitutively active Rho forms

CDNA for the candidate Rho GTPase is cloned from a rat tufted cDNA library, and a mutation is introduced that creates a D / N or C / A form of a Rho protein (Qui RG et al, Proc Natl Acad Sci USA 5 Dec 1995; 92 (25): 11781-5; Lu X et al, Curr Biol. 1 Dec 1996; 6 (12): 1677-84), and each cDNA is added a sequence that encodes a marker of myc. A recombinant adenovirus that expresses mutant Rho family proteins is created and used to overexpress the mutant proteins in CMMh.

RESULTS

To further study the role of Rho family proteins in the transmission of information about the extracellular matrix, activities of Rho family proteins in CMM cultured in soft or nested gels are tested. Additional Rho family proteins involved in the transmission of these signals are identified.

In additional experiments, the dominant negative form (D / N) or the constitutively active form (C / A) of a Rho family of interest protein is overexpressed in CMMh. Recombinant adenovirus is used that expresses

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LacZ as a negative control. CMMh of soft gels, neat gels or glass coverslips are prepared, incubated for 24 hours, then left uninfected or infected with adenovirus expressing LacZ or mutant forms of Rho family proteins. After 36 hours of incubation, BrdU is added to the medium, and the cells are incubated for an additional 12 hours in serum-containing medium, then fixed and immunostained with respect to myc and BrdU marker using anti myc antibodies (Cell Signaling Technology , Danvers, MA) and anti BrdU antibodies (Invitrogen, Carlsbad, CA). Comparison of the number of positive cells for staining with BrdU between uninfected cells and cells infected with adenovirus LacZ is used to confirm that adenovirus infection itself has no effect on the growth of CMMh. The number of positive cells for BrdU incorporation is compared with the positive number for staining with myc marker. The negative modulation of growth arrest induced by soft matrix or promotion of growth induced by matrix defined by C / A and D / N forms of Rho family proteins, respectively, indicates involvement of the Rho family protein overexpressed in the CMMh growth regulation by matrix stiffness.

Example 12: Determination of the role of actomyosin in the regulation of stem cell growth

To study the role of actomyosin in the regulation of the growth of CMMh in soft gels before committing to specific cell lineages, the CMMh are seeded in glass coverslips coated with a mixture of type I collagen and fibronectin for 24 hours, after which incubate with BrdU in the presence or absence of 20 mM 2,3-butanedione monoxime (BDM), 100 pM blebistatin or 0.25 pM / ml cytochalasin D (CD) for an additional 12 hours. Throughout the experiment, the cells are incubated in the presence of serum. After incubation, the cells are fixed and immunostained with respect to BrdU. The effect on the growth of CMMh of myosin II inhibition by BDM or blebistatin, or the alteration of actin filaments by CD is evaluated.

Example 13: growth and differentiation of CMMh in soft three-dimensional fibrin gels

EXPERIMENTAL MATERIALS AND METHODS

Repair and planting of fibrin gels

Salmon fibrinogen (Searun Holdings, Freeport, ME) is rehydrated in H2O and diluted to 3 (for soft gel) or 18 mg / ml (for nested gel) in 50 mM Tris, 150 mM NaCl, pH 7.4 and polymerize almuots of 400 microliters (pl) with 2 units / ml of fish thrombin (Searun Holdings) in tissue culture wells. The stiffness of salmon fibrin gels prepared from fibrinogen 3 and 18 mg / ml is 250 Pa and 2150 Pa, respectively. Prior to polymerization, 104 CMMh is mixed with fibrinogen solution in dMeM containing 10% FCS, in which the cells are incubated for 24 hours.

RESULTS

The CMM proliferation analysis in soft gels and fibrin nests is evaluated by incubating cells for an additional 12 hours in the presence of serum and BrdU, followed by fixation and immunostaining for BrdU incorporation.

The strength of CMMh differentiation in fibrin gels is assessed through the efficacy of adipocyte differentiation. The differentiation of CMM in fibrin gels in adipocytes is induced by changing the medium to Adipogenic Induction Medium (“MIA” DMEM + 10% FBS, 1 micromolar dexamethasone (pM), indomethacin 200 pM, insulin 10 micrograms (pg) / ml and 0.5 mM methyl isobutylxanthine) for 3 days, then keeping the cells in Adipogenic Maintenance Medium (MC, insulin 10 pg / ml). 8 days after switching to Adipogenic Induction Medium, the differentiation of adipocytes is evaluated either by staining with Oil Red O or by anti-PPARy2 immunostaining. The percentages of positive cells for staining with Oil Red O or staining with PPARy2 are compared between soft and nigid fibrin gels.

In other experiments, protease inhibitors are added to the matrix to prevent or inhibit proteolytic degradation or other active remodeling of the cells.

Example 14: use of CMMh of the present invention to maintain the viability of hematopoietic stem cells

Pass CMM suspensions 10 are seeded in 96-well tissue culture plates coated with type I collagen plus fibronectin at a density of 103 cells / well. After incubating the cells in MC for 24 hours, the cells are cultured in the presence of a soft gel or a soft matrix, as described in the previous Examples. The resulting mesenchymal stem cell cultures are then added to hematopoietic stem cell cultures to maintain the viability of these latter cells.

In additional studies, CMMh are incubated directly in the soft gel, without prior seeding in tissue culture plates and trypsinization.

Having described the preferred embodiments of the invention with reference to the accompanying drawings, it should be understood that the invention is not limited to specific embodiments and that those skilled in the art can make various changes and modifications therein without departing from the scope of the invention as defined in the appended claims.

Claims (7)

5 10 fifteen twenty 25 30 35 40 1. A method of inducing the differentiation of a population of quiescent mesenchymal stem cells in a population of a type of cell of interest, said method comprising the steps of (a) maintaining said population of quiescent mesenchymal stem cells, preserving the capacity that said population of mesenchymal stem cells differs in multiple types of cells and preserving the proliferative capacity of said population of mesenchymal stem cells in the presence of an apparatus containing a gel or a gel matrix having a shear modulus in an interval from 150-750 Pa; and (b) cultivating said population of mesenchymal stem cells in the presence of an induction medium, thereby inducing the differentiation of said population of mesenchymal stem cells in a population of a type of cell of interest. 2. The method of claim 1, wherein said population of a type of cell of interest is a population of adipocytes. 3. The method of claim 1, wherein said population of a type of cell of interest is a population of osteoblasts. 4. The method of claim 1, wherein (a) said gel or gel matrix further comprises an animal serum; I (b) the maintenance stage of said population of mesenchymal stem cells in a gel or a gel matrix is preceded by a culture stage of said population of mesenchymal stem cells in a tissue culture apparatus; I (c) the maintenance stage is performed directly after isolation, purification or enrichment of said population of mesenchymal stem cells from a biological sample. 5. The method of claim 1, wherein the induction medium is an adipogenic induction medium. 6. The method of claim 1, wherein a) said gel or gel matrix comprises acrylamide and bisacrylamide; I b) said gel or the gel matrix is two-dimensional or three-dimensional; I c) said gel or gel matrix is coated with an adhesion protein; I d) said population of mesenchymal stem cells is a population of adult mesenchymal stem cells; I e) said population of mesenchymal stem cells is a population of human mesenchymal stem cells. 7. The method of claim 6, wherein (a) said gel or gel in said gel matrix has a total acrylamide concentration of 3% and a total bisacrylamide concentration of 0.06% to 0.5%, a total acrylamide concentration of 5.5% and a total bisacrylamide concentration of 0.05% to 0.075% or a total acrylamide concentration of 7.5% and a total bisacrylamide concentration of 0.01% to 0.03%; I (b) said adhesion protein is a collagen, a fibronectin, or a combination thereof.